Backbone modified oligonucleotide analogues

ABSTRACT

Therapeutic oligonucleotide analogues which have improved nuclease resistance and improved cellular uptake are provided. Replacement of the normal phosphorodiester inter-sugar linkages found in natural oligomers with four atom linking groups forms unique di- and poly-nucleosides and nucleotides useful in regulating RNA expression and in therapeutics. Methods of synthesis and use are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of Ser. No. 09/058,470, filed Apr.10, 1998 now abandoned which in turn is a divisional of Ser. No.08/763,354 (now U.S. Pat. No. 5,965,721) filed Dec. 11, 1996 which is adivisional of Ser. No. 08/150,079 (now U.S. Pat. No. 5,610,289) fileApr. 7, 1994 which is a 371 of PCT/US92/04294, filed May 21, 1992 whichis a continuation-in-part of Ser. No. 07/703,619 (now U.S. Pat. No.5,378,825) filed May 21, 1991 which is a continuation-in-part of Ser.No. 07/566,836 (now U.S. Pat. No. 5,223,618) filed Aug. 13, 1990 whichis a continuation-in-part of Ser. No. 07/558,663 (now U.S. Pat. No.5,138,045) filed Jul. 27, 1990), the entirety of which is incorporatedherein by reference.

FIELD OF THE INVENTION

This invention relates to the design, synthesis and application ofnuclease resistant oligonucleotide analogues which are useful fortherapeutics, diagnostics and as research reagents. Oligonucleotideanalogues are provided that have modified linkages which replacephosphorodiester bonds which normally serve as inter-sugar linkages innatural nucleic acids. Such analogues are resistant to nucleasedegradation and are capable of modulating the activity of DNA and RNA.Methods for synthesizing these oligonucleotide analogues and formodulating the production of proteins, utilizing the oligonucleotideanalogues of the invention are also provided as are intermediatecompositions and methods.

BACKGROUND OF THE INVENTION

It is well known that most of the bodily states in mammals includingmost disease states, are effected by proteins. Such proteins, eitheracting directly or through their enzymatic functions, contribute inmajor proportion to many diseases in animals and man.

Classical therapeutics has generally focused upon interactions with suchproteins in an effort to moderate their disease causing or diseasepotentiating functions. Recently, however, attempts have been made tomoderate the actual production of such proteins by interactions with themolecules, i.e. intracellular RNA, that direct their synthesis. Theseinteractions have involved the hybridization of complementary“antisense” oligonucleotides or certain analogues thereof to RNA.Hybridization is the sequence-specific hydrogen bonding ofoligonucleotides or oligonucleotide analogues to RNA or single strandedDNA. By interfering with the production of proteins, it has been hopedto effect therapeutic results with maximum effect and minimal sideeffects. In the same way, oligonucleotide analogues may modulate theproduction of proteins by an organism.

The pharmacological activity of antisense oligonucleotides andoligonucleotide analogues, like other therapeutics, depends on a numberof factors that influence the effective concentration of these agents atspecific intracellular targets. One important factor foroligonucleotides is the stability of the species in the presence ofnucleases. It is unlikely that unmodified oligonucleotides will beuseful therapeutic agents because they are rapidly degraded bynucleases. Modifications of oligonucleotides to render them resistant tonucleases is therefore greatly desired.

Modifications of oligonucleotides to enhance nuclease resistance havegenerally taken place on the phosphorus atom of the sugar-phosphatebackbone. Phosphorothioates, methyl phosphonates, phosphoramidates andphosphotriesters have been reported to confer various levels of nucleaseresistance; however, the phosphate modified oligonucleotides havegenerally suffered from inferior hybridization properties. Cohen, J. S.,ed. Oligonucleotides: Antisense Inhibitors of Gene Expression, (CRCPress, Inc., Boca Raton Fla., 1989).

Another key factor is the ability of antisense compounds to traverse theplasma membrane of specific cells involved in the disease process.Cellular membranes consist of lipid-protein bilayers that are freelypermeable to small, nonionic, lipophilic compounds and inherentlyimpermeable to most natural metabolites and therapeutic agents. Wilson,D. B. Ann. Rev. Biochem. 47:933-965 (1978). The biological and antiviraleffects of natural and modified oligonucleotides in cultured mammaliancells have been well documented, thus it appears that these agents canpenetrate membranes to reach their intracellular targets. Uptake ofantisense compounds into a variety of mammalian cells, including HL-60,Syrian Hamster fibroblast, U937, L929, CV-1 and ATH8 cells has beenstudied using natural oligonucleotides and certain nuclease resistantanalogues, such as alkyl triesters, Miller, P. S., Braiterman, L. T. andTs'O, P. O. P., Biochemistry 16:1988-1996 (1977); methyl phosphonates,Marcus-Sekura, C. H., Woerner, A. M., Shinozuka, K., Zon, G., andQuinman, G. V., Nuc. Acids Res. 15:5749-5763 (1987) and Miller, P. S.,McParland, K. B., Hayerman, K. and Ts'O, P. O. P., Biochemistry 16:1988-1996 (1977) and Loke, S. K., Stein, C., Zhang, X. H. Avigan, M.,Cohen, J. and Neckers, L. M. Top. Microbiol. Immunol. 141: 282:289(1988).

Often, modified oligonucleotide and oligonucleotide analogues are lessreadily internalized than their natural counterparts. As a result, theactivity of many previously available antisense oligonucleotides has notbeen sufficient for practical therapeutic, research or diagnosticpurposes. Two other serious deficiencies of prior art oligonucleotidesthat have been designed for antisense therapeutics are inferiorhybridization to intracellular RNA and the lack of a defined chemical orenzyme-mediated event to terminate essential RNA functions.

Modifications to enhance the effectiveness of the antisenseoligonucleotides and overcome these problems have taken many forms.These modifications include base ring modifications, sugar moietymodifications and sugar-phosphate backbone modifications. Priorsugar-phosphate backbone modifications, particularly on the phosphorusatom, have effected various levels of resistance to nucleases. However,while the ability of an antisense oligonucleotide to bind to specificDNA or RNA with fidelity is fundamental to antisense methodology,modified phosphorus oligonucleotides have generally suffered frominferior hybridization properties.

Replacement of the phosphorus atom has been an alternative approach inattempting to avoid the problems associated with modification on thepro-chiral phosphate moiety. Some modifications in which replacement ofthe phosphorus atom has been achieved are; Matteucci, M. TetrahedronLetters 31:2385-2388 (1990), wherein replacement of the phosphorus atomwith a methylene group is limited by available methodology which doesnot provide for uniform insertion of the formacetal linkage throughoutthe backbone, and its instability, making it unsuitable for work;Cormier, et al. Nucleic Acids Research 16:4583-4594 (1988), whereinreplacement of the phosphorus moiety with a diisopropylsilyl moiety islimited by methodology, solubility of the homopolymers and hybridizationproperties; Stirchak, et al. Journal of Organic Chemistry 52:4202-4206(1987) wherein replacement of the phosphorus linkage by shorthomopolymers containing carbamate or morpholino linkages is limited bymethodology, the solubility of the resulting molecule, and hybridizationproperties; Mazur, et al. Tetrahedron 40:3949-3956 (1984) whereinreplacement of the phosphorus linkage with a phosphonic linkage has notbeen developed beyond the synthesis of a homotrimer molecule; andGoodchild, J., Bioconjugate Chemistry 1:165-187 (1990) wherein esterlinkages are enzymatically degraded by esterases and are thereforeunsuitable to replace the phosphate bond in antisense applications.

The limitations of the available methods for modification of thephosphorus backbone have led to a continuing and long felt need forother modifications which provide resistance to nucleases andsatisfactory hybridization properties for antisense oligonucleotidediagnostics, therapeutics, and research.

OBJECTS OF THE INVENTION

It is an object of the invention to provide oligonucleotide analoguesfor use in antisense oligonucleotide diagnostics, research reagents, andtherapeutics.

It is a further object of the invention to provide oligonucleotideanalogues which possess enhanced cellular uptake.

Another object of the invention is to provide such oligonucleotideanalogues which have greater efficacy than unmodified antisenseoligonucleotides.

It is yet another object of the invention to provide methods forsynthesis and use of such oligonucleotide analogues.

These and other objects will become apparent to persons of ordinaryskill in the art from a review of the present specification and theappended claims.

SUMMARY OF THE INVENTION

Compositions useful for modulating the activity of an RNA or DNAmolecule in accordance with this invention generally compriseoligonucleotide analogues having at least portions of their backbonelinkages modified. In these modifications the phosphorodiester linkageof the sugar phosphate backbone found in natural nucleic acids has beenreplaced with various four atom linking groups. Such four atom linkinggroups maintain a desired four atom spacing between the 3′-carbon of onesugar or sugar analogue and the 4′-carbon of the adjacent sugar or sugaranalogue. Oligonucleotide analogues made in accordance with theteachings of the invention are comprised of a selected sequence which isspecifically hybridizable with a preselected nucleotide sequence ofsingle stranded or double stranded DNA or RNA. They are synthesizedconveniently, through known solid state synthetic methodology, to becomplementary to or at least to be specifically hybridizable with thepreselected nucleotide sequence of the RNA or DNA. Nucleic acidsynthesizers are commercially available and their use is generallyunderstood by persons of ordinary skill in the art as being effective ingenerating nearly any oligonucleotide or oligonucleotide analogue ofreasonable length which may be desired.

In the context of this invention, the term “nucleoside” as the term isused in connection with this invention refers to the unit made up of aheterocyclic base and its sugar. The term “nucleotide” refers to anucleoside having a phosphate group on its 3′ or 5′ sugar hydroxylgroup. Thus nucleosides, unlike nucleotides, have no phosphate group.“Oligonucleotide” refers to a plurality of joined nucleotide unitsformed in a specific sequence from naturally occurring bases andpentofuranosyl groups joined through a sugar group by nativephosphodiester bonds. These nucleotide units may be nucleic acid basessuch as guanine, adenine, cytosine, thymine or uracil. The sugar groupmay be a deoxyribose or ribose. This term refers to both naturallyoccurring and synthetic species formed from naturally occurringsubunits.

“Oligonucleotide analogue” as the term is used in connection with thisinvention, refers to moieties which function similarly tooligonucleotides but which have non-naturally occurring portions.oligonucleotide analogues may have altered sugar moieties, altered basemoieties or altered inter-sugar linkages. For the purposes of thisinvention, an oligonucleotide analogue having non-phosphodiester bonds,i.e. an altered inter-sugar linkage, can alternately be considered as an“oligonucleoside.” Such an oligonucleoside thus refers to a plurality ofjoined nucleoside units joined by linking groups other than nativephosphodiester linking groups. Additionally for the purposes of thisinvention the terminology “oligomers” can be considered to encompassoligonucleotides, oligonucleotide analogues or oligonucleosides. Thus inspeaking of “oligomers” reference is made to a series of nucleosides ornucleoside analogues that are joined together via either naturalphosphodiester bonds or via other linkages including the four atomlinkers of this invention. Generally while the linkage is from the 3′carbon of one nucleoside to the 5′ carbon of a second nucleoside, theterm “oligomer” can also include other linkages such as a 2′-5′ linkage.

Oligonucleotide analogues may also comprise other modificationsconsistent with the spirit of this invention, and in particular suchmodifications as may increase nuclease resistance of the oligonucleotidecomposition in order to facilitate antisense therapeutic, diagnostic, orresearch reagent use of a particular oligonucleotide. For example, whenthe sugar portion of a nucleoside or nucleotide is replaced by acarbocyclic or other moiety, it is no longer a sugar. Moreover, whenother substitutions, such a substitution for the inter-sugarphosphorodiester linkage are made, the resulting material is no longer atrue nucleic acid species. All such are denominated as analogues,however. Throughout this specification, reference to the sugar portionof a nucleic acid species shall be understood to refer to either a truesugar or to a species taking the traditional space of the sugar ofnatural nucleic acids. Moreover, reference to inter-sugar linkages shallbe taken to include moieties serving to join the sugar or sugar analogueportions together in the fashion of natural nucleic acids.

In accordance with the present invention, novel types of antisenseoligonucleotide analogues and oligonucleosides are provided which aremodified to enhance cellular uptake, nuclease resistance, andhybridization properties and to provide a defined chemical orenzymatically mediated event to terminate essential RNA functions.

It has been found that certain classes of oligonucleotide analoguecompositions can be useful in therapeutics and for other objects of thisinvention. Such oligonucleotide analogues are comprised of subunits, atleast some of which have the structure:

wherein B_(x) is a variable base moiety; Q is O, CH₂, CHF or CF₂ and Xis H; OH; C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl oraralkyl; F; Cl; Br; CN; CF₃; OCF₃; OCN; O—, S—, or N-alkyl; O—, S—, orN-alkenyl; SOCH₃; SO₂CH₃; ONO₂; NO₂; N₃; NH₂; heterocycloalkyl;heterocycloalkaryl; aminoalkylamino; polyalkylamino or substitutedsilyl. Moreover, X can be an RNA cleaving group; a group for improvingthe pharmacokinetic properties of an oligonucleotide; or a group forimproving the pharmacodynamic properties of an oligonucleotide.

L₁ and L₄ are, independently, CH₂, C═O, C═S, C—NH₂, C—NHR₃, C—OH, C—SH,C—O—R, or C—S—R₁. L₂ and L₃ are, independently, CR₁R₂, C═CR₁R₂, C═NR₃,P(O)R₄, P(S)R₄, C═O, C═S, O, S, SO, SO₂, NR₃ or SiR₅R₆; or together,form part of an alkene, alkyne, aromatic ring, carbocycle orheterocycle. L₁, L₂, L₃ and L₄ are as noted with the proviso that if L₁is C═O or C═S then L₂ is not NR₃ or if L₄ in C═O or C═S then L₃ is notNR₃. Further L₁, L₂, L₃ and L₄, together, may comprise a —CH═N—NH—CH₂—or —CH₂—O—N═CH— moiety.

R₁ and R₂ are, independently, H; OH; SH; NH₂; C₁ to C₁₀ alkyl,substituted alkyl, alkenyl, alkaryl or aralkyl; alkoxy; thioalkoxy;alkylamino; aralkylamino; substituted alkylamino; heterocycloalkyl;heterocycloalkylamino; aminoalkylamino; polyalkylamino; halo; formyl;keto; benzoxy; carboxamido; thiocarboxamido; ester; thioester;carboxamidine; carbamyl; ureido or guanidino. They may alsoindependently comprise an RNA cleaving group; a group for improving thepharmacokinetic properties of an oligonucleotide; or a group forimproving the pharmacodynamic properties of an oligonucleotide;

R₃ is H, OH, NH₂, lower alkyl, substituted lower alkyl, alkoxy, loweralkenyl, aralkyl, alkylamino, aralkylamino, substituted alkylamino,heterocycloalkyl, heterocycloalkylamino, aminoalkylamino,polyalkylamino, an RNA cleaving group, a group for improving thepharmacokinetic properties of an oligonucleotide or a group forimproving the pharmacodynamic properties of an oligonucleotide. R₄ isOH, SH, NH₂, O-alkyl, S-alkyl, NH-alkyl, O-alkylheterocyclo,S-alkylheterocyclo, N-alkylheterocyclo or a nitrogen-containingheterocycle.

R₅ and R₆ are, independently, C₁ to C₆ alkyl or alkoxy; provided that ifL₂ is P(O)R₄ and R₄ is OH and X is OH and B_(x) is uracil or adenine,then L₃ is not O; and that if L₁, L₂ and L₄ are CH₂ and X is H or OH andQ is O then L₃ is not S, SO or SO₂.

In accordance with preferred embodiments, the oligonucleotide analoguesof the invention comprise sugar moieties, such that Q is O. Inaccordance with other embodiments, each of L₁ and L₄ is CR₁R₂. It isalso preferred that L₂ and L₃ be, independently, CR₁R₂, O, P(O)R₄,P(S)R₄ or NR₃ and especially that one of L₂ and L₃ be CR₁R₂ and theother of L₂ and L₃ be P(O)R₄ or P(S)R₄. Combinations where L₂ is O andL₃ is P(O)R₄ or P(S)R₄ are also preferred.

In accordance with other preferred embodiments, the oligonucleotideanalogues of this invention are such that each of L₂ and L₃ is NR₃ whereR₃ is preferably H. Alternatively, the analogues of the invention may besuch that L₂ and L₃, taken together, form a portion of a cyclopropyl,cyclobutyl, ethyleneoxy, ethyl aziridine or substituted ethyl aziridinering. L₂ and L₃ taken together may also form a portion of a C₃ to C₆carbocycle or 4-, 5- or 6-membered nitrogen heterocycle.

It is preferred that the oligonucleotide analogues be such that X is Hor OH, or, alternatively F, O-alkyl or O-alkenyl, especially where Q isO. The group B_(x) is preferably adenine, guanine, uracil, thymine,cytosine, 2-aminoadenosine or 5-methylcytosine, although othernon-naturally occurring species may be employed.

Other preferred embodiments are those where L₁ and L₄ are each CH₂,especially where L₂ and L₃ are each NH. Alternatively, one of L₂ and L₃,preferably, L₃, is O and the other of L₂ and L₃ is NH.

It is preferred that the oligonucleotide analogues of the inventioncomprise from about 5 to about 50 subunits having the given structure.While substantially each subunit of the oligonucleotide analogues mayhave said structure, it is also desirable for substantially alternatingsubunits to have said structure.

The oligonucleotide analogues of this invention are preferably preparedin a pharmaceutically acceptable carrier for therapeutic administrationto patients. The analogues are believed to exhibit improved nucleaseresistance as compared to corresponding natural oligonucleotides.

This invention is also directed to methods for modulating the productionor activity of a protein in an organism comprising contacting theorganism with an oligonucleotide analogue specifically hybridizable withat least a portion of a nucleic acid sequence coding for said protein,wherein at least some of the subunits of the analogue have the foregoingstructure.

Additionally, the invention is directed to methods for treating anorganism having a disease characterized by the undesired production of aprotein comprising contacting the organism with an oligonucleotideanalogue hybridizable with at least a portion of a nucleic acid sequencecoding for said protein, either alone or in a pharmaceuticallyacceptable carrier, wherein at least some of the subunits of theanalogue have the given structure.

This invention also provides methods for synthesizing oligonucleotideanalogues including those useful in the practice of the therapeuticmethods of the invention comprising providing a first moiety comprisingthe structure:

and a second moiety comprising the structure:

wherein B_(x) is a variable base moiety; Q is O, CH₂, CHF or CF₂; and E₁and E₂ are the same or different and are electrophilic reactive groups;and coupling said first and second moieties with a linking group throughsaid electrophilic reactive groups to form said oligonucleotideanalogue. In accordance with preferred methods, the electrophilicreactive group of the first moiety comprises halomethyl,trifluoromethyl, sulfonylmethyl, p-methyl-benzene sulfonyl-methyl, or3′-C-formyl, while the electrophilic reactive group of the second moietycomprises halogen, sulfonylmethyl, p-methyl-benzene sulfonyl methyl, oraldehyde. It is preferred that the linking group be hydrazine orhydroxylamine.

The invention also provides a method of protecting the L₂ or L₃ nitrogenmoiety of an oligonucleotide analogue as described above wherein one ofL₂ or L₃ is NR₃ and R₃ is H. This method includes blocking the nitrogenmoiety with phenoxyacetylchloride, further reacting the oligonucleotideanalogue to modify the oligonucleotide and deblocking the nitrogenmoiety with ammonium hydroxide.

The invention also provides a method of protecting a bifunctionalnucleoside or oligonucleotide analogue wherein one of thebifunctionalities is an aldehyde. This method includes reacting thealdehyde with methoxyamine to form an oxime derivative of the aldehyde,further reacting the nucleoside or oligonucleoside analogue to modifythe nucleoside or oligonucleotide analogue and reacting the oxime withan acetaldehyde to regenerate the aldehyde.

The invention also provides a method of synthesizing an oligonucleotideanalogue as described above wherein the method includes generating aradical at the 3′ carbon atom of a pentofuranosyl nucleoside andreacting that radical with an oxime moiety that is pendent on the 5′position of a further pentofuranosyl nucleoside.

It is useful to formulate therapeutic compositions where at least oneportion of said oligonucleotide analogue is incorporated into a furtheroligonucleotide species to provide said further oligonucleotide analoguewith natural phosphodiester bonds substantially alternating with areasso coupled. The incorporation is preferably achieved by phosphodiesterlinkage of a desired sequence of dinucleotides, said dinucleotideshaving been previously so coupled.

Precursor nucleosides are also contemplated by this invention having thestructure:

wherein B_(x) is a variable base moiety Q is O, CH₂, CHF or CF₂; and Xis H; OH; C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl oraralkyl; F; Cl; Br; CN; CF₃; OCF₃; OCN; O—, S—, or N-alkyl; O—, S—, orN-alkenyl; SOCH₃; SO₂CH₃; ONO₂; NO₂; N₃; NH₂; heterocycloalkyl;heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl;an RNA cleaving group; a group for improving the pharmacokineticproperties of an oligonucleotide; or a group for improving thepharmacodynamic properties of an oligonucleotide.

In such species, Y is hydroxyl, aminomethyl, hydrazinomethyl,hydroxymethyl, C-formyl, phthalimidohydroxymethyl, aryl-substitutedimidazolidino, aminohydroxylmethyl, ortho-methylaminobenzenethio,methylphosphonate and methyl-alkylphosphonate. Z is H, hydroxyl,aminomethyl, hydrazinomethyl, hydroxymethyl, C-formyl,phthalimidohydroxymethyl, aryl substituted imidazolidino,aminohydroxylmethyl, ortho-methylaminobenzenethio, methylphosphonate ormethyl alkylphosphonate.

All of the foregoing is with the proviso that when Q is O and Y ishydroxymethyl and X is H or OH then Z is not H or C-formyl; and thatwhen Q is O and X is H or OH and Z is hydroxyl then Y is notaminohydroxylmethyl, hydrazinomethyl or aryl-substituted imidazolidino.It is preferred that X be H or OH and that Q be O.

Oligonucleotide analogues having modified sugar linkages have been foundto be effective in accomplishing these goals. The oligonucleotideanalogues may preferably range in size from about 5 to about 50 nucleicacid base subunits in length. Oligonucleotide analogues described inthis invention are hybridizable with preselected nucleotide sequences ofsingle stranded or double stranded DNA and RNA. The nucleic acid baseswhich comprise this invention may be pyrimidines such as thymine, uracilor cytosine or purines such as guanine or adenine, or modificationsthereof such as 5-methylcytosine, arranged in a selected sequence. Thesugar moiety may be of the ribose or deoxyribose type or a sugar mimicsuch as a carbocyclic ring. In accordance with one preferred embodimentof this invention, the oligonucleotide analogues or oligonucleosideshybridize to HIV mRNA encoding the tat protein, or to the TAR region ofHIV mRNA. In another preferred embodiment, the oligonucleotide analoguesor oligonucleosides mimic the secondary structure of the TAR region ofHIV mRNA, and by doing so bind the tat protein. Other preferredantisense oligonucleotide analogue or oligonucleoside sequences includecomplementary sequences for herpes, papilloma and other viruses.

The modified linkages of this invention preferably are comprised of afour atom linking group to replace the naturally occurringphosphodiester-5′-methylene linkage. Replacement of the naturallyoccurring linkage by four atom linkers of the present invention confersnuclease resistance and enhanced cellular uptake upon the resultingoligonucleotide analogue. Included within the four atom linker ispreferably a 3′-deoxy function on one of the linked sugars. The fouratom linker is of the structure —L₁—L₂—L₃—L₄— wherein L₁ and L₄ aremethylene carbon atoms or substituted carbon atoms and L₂ and L₃ aremethylene carbon atoms, substituted carbon atoms, oxygen atoms, nitrogenor substituted nitrogen atoms, substituted phosphorus atoms, sulfur orsubstituted sulfur atoms or substituted silicon atoms. It is preferredthat the modified linkage occur at substantially each linkage location.Alternatively, modification may occur at less than every location suchas at alternating linkage locations. The linkage may be neutral or maybe positively or negatively charged.

This invention is also directed to methods for synthesizing sucholigonucleosides. The invention provides for the coupling of a3′-deoxy-3′-substituted, especially methyl substituted, nucleoside witha 5′-deoxy-5′-substituted nucleoside through the addition of a two atomfragment or substituted two atom fragment. The addition reaction mayoccur through a stepwise procedure involving the activation of the 3′and 5′ positions of respective nucleosides to a variety of suitableelectrophilic moieties, followed by the addition of a suitable linkinggroup to react with the electrophiles. In the alternative, the proceduremay occur in a concerted manner. Such methods may employ solid supportsvia a DNA synthesizer, by manual manipulation of the support, orotherwise.

This invention is also directed to methods for modulating the productionof proteins by an organism comprising contacting the organism with acomposition formulated in accordance with the foregoing considerations.It is preferred that the RNA or DNA portion which is to be modulated bepreselected to comprise that portion of DNA or RNA which codes for theprotein whose formation or activity is to be modulated. The targetingportion of the composition to be employed is, thus, selected to becomplementary to the preselected portion of DNA or RNA, that is to be anantisense oligonucleotide for that portion.

This invention is also directed to methods for treating an organismhaving a disease characterized by the undesired production of a protein.This method comprises contacting the organism with a composition inaccordance with the foregoing considerations. The composition ispreferably one which is designed to specifically bind with messenger RNAwhich codes for the protein whose production or activity is to bemodulated. The invention further is directed to diagnostic methods fordetecting the presence or absence of abnormal RNA molecules or abnormalor inappropriate expression of normal RNA molecules in organisms orcells.

This invention is also directed to methods for the selective binding ofRNA for research and diagnostic purposes. Such selective, strong bindingis accomplished by interacting such RNA or DNA with compositions of theinvention which are resistant to degradative nucleases and whichhybridize more strongly and with greater fidelity than knownoligonucleotides or oligonucleotide analogues.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic, synthetic scheme in accordance with certainembodiments of the invention; and

FIG. 2 is a schematic, synthetic scheme in accordance with furtherembodiments of the invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The biological activity of the antisense oligonucleotides previouslyavailable has not generally been sufficient for practical therapeuticresearch or diagnostic use. This invention directs itself to modifiedoligonucleotides, i.e. oligonucleotide analogues or oligonucleosides,and methods for effecting such modifications. These modifiedoligonucleotides and oligonucleotide analogues exhibit increasedstability relative to their naturally occurring counterparts.Extracellular and intracellular nucleases generally do not recognize andtherefore do not bind to the backbone modified oligonucleotide analoguesor oligonucleosides of the present invention. Any binding by a nucleaseto the backbone will not result in cleavage of the nucleosidic linkagesdue to the lack of sensitive phosphorus-oxygen bonds. In addition, the,resulting, novel neutral or positively charged backbones of the presentinvention may be taken into cells by simple passive transport ratherthan requiring complicated protein mediated processes. Another advantageof the present invention is that the lack of a negatively chargedbackbone facilitates the sequence specific binding of theoligonucleotide analogues or oligonucleosides to targeted RNA, which hasa negatively charged backbone, and which will accordingly repel incomingsimilarly charged oligonucleotides. Still another advantage of thepresent invention is that sites for attaching functional groups whichcan initiate catalytic cleavage of targeted RNA are found in thesestructure types.

In accordance with preferred embodiments, this invention is directed toreplacing inter-sugar phosphate groups to yield analogues havinglinkages as found in the structure:

wherein

-   -   B_(x) is a variable base moiety;    -   Q is O, CH₂, CHF or CF₂;    -   X is H; OH; C₁ to C₁₀ lower alkyl, substituted lower alkyl,        alkaryl or aralkyl; F; Cl; Br; CN; CF₃; OCF₃; OCN; O—, S—, or        N-alkyl; O—, S—, or N-alkenyl; SOCH₃; SO₂CH₃; ONO₂; NO₂; N₃;        NH₂; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino;        polyalkylamino; substituted silyl; an RNA cleaving group; a        group for improving the pharmacokinetic properties of an        oligonucleotide; or a group for improving the pharmacodynamic        properties of an oligonucleotide;    -   L₁ and L₄ are, independently, CH₂, C═O, C═S, C—NH₂, C—NHR₃,        C—OH, C—SH, C—O—R₁ or C—S—R₁; and    -   L₂ and L₃ are, independently, CR₁R₂, C═CR₁R₂, C═NR₃, P(O)R₄,        P(S)R₄, C═O, C═S, O, S, SO, SO₂, NR₃ or SiR₅R₆; or, together,        form part of an alkene, alkyne, aromatic ring, carbocycle or        heterocycle; or    -   L₁, L₂, L₃ and L₄, together, comprise a —CH═N—NH—CH₂— or        —CH₂—O—N═CH— moiety;    -   R₁ and R₂ are, independently, H; OH; SH; NH₂; C₁ to C₁₀ alkyl,        substituted alkyl, alkenyl, alkaryl or aralkyl; alkoxy;        thioalkoxy; alkylamino; aralkylamino; substituted alkylamino;        heterocycloalkyl; heterocycloalkylamino; aminoalkylamino;        polyalkylamino; halo; formyl; keto; benzoxy; carboxamido;        thiocarboxamido; ester; thioester; carboxamidine; carbamyl;        ureido; guanidino; an RNA cleaving group; a group for improving        the pharmacokinetic properties of an oligonucleotide; or a group        for improving the pharmacodynamic properties of an        oligonucleotide;    -   R₃ is H, OH, NH₂, lower alkyl, substituted lower alkyl, alkoxy,        lower alkenyl, aralkyl, alkylamino, aralkylamino, substituted        alkylamino, heterocycloalkyl, heterocycloalkylamino,        aminoalkylamino, polyalkylamino, an RNA cleaving group, a group        for improving the pharmacokinetic properties of an        oligonucleotide and a group for improving the pharmacodynamic        properties of an oligonucleotide;    -   R₄ is OH, SH, NH₂, O-alkyl, S-alkyl, NH-alkyl,        O-alkylheterocycle, S-alkylheterocycle, N-alkylheterocycle or a        nitrogen-containing heterocycle; and    -   R₅ and R₆ are, independently, C₁ to C₆ alkyl or alkoxy;        provided that if L₁ is C═O or C═S then L₂ is not NR₃ or if L₄ is        C═O or C═S then L₃ is not NR₃; and that if one of L₂ or L₃ is        C═O or C═S then the other of L₂ or L₃ is not NR₃; and that if L₂        is P(O)R₄ and R₄ is OH and X is OH and B_(x) is uracil or        adenine, then L₃ is not O; and that if L₁, L₂ and L₄ are CH₂ and        X is H or OH and Q is O then L₃ is not S, SO or SO₂.

In accordance with preferred embodiments of the invention L₁ and L₄ aremethylene groups. In such preferred embodiments one of L₂ or L₃ cancomprise an amino group and the other comprise an amino group or anoxygen. Thus in certain preferred embodiments L₂ and L₃ together arehydrazino, aminohydroxy or hydroxyamino. In other preferred embodimentsone of L₁ or L₄ together with one of L₂ or L₃ are a CH═N group and theother of L₂ or L₃ is an oxygen or nitrogen atom thus the linker includesoxime and hydrazone groupings, respectively. Such oxime or hydrazonelinking groups can be reduced to the above referenced aminohydroxy orhydrazine groups.

In other preferred embodiments of the present invention, L₂ and L₃ aresubstituted carbon, amino, substituted amine, oxygen, sulfur, oxides ofsulfur, phosphorus or silicon. The substituents on carbon includehydrogen, hydroxy, thio, amino, lower alkyl, substituted lower alkyl,alkoxy, thioalkoxy, lower alkenyl, aralkyl, alkylamino, aralkylamino,substituted alkylamino, heterocycloalkyl, heterocycloalkylamino,aminoalkylamino, polyalkylamino, halogen, formyl, keto, benzoxy, ester,thioester, carboxamidine, guanidino, an RNA cleaving group, a group forimproving the pharmacokinetic properties of an oligonucleotide or agroup for improving the pharmacodynamic properties of anoligonucleotide. Additional preferred embodiments include L₂ and L₃together being C═C. Further preferred embodiments include L₂ and L₃together being a C—C, C═C, C—N or N—C two atom pair of a ring structureincluding carbocyclic, aromatic, heteroaromatic or heterocyclic rings.Still another preferred embodiment of the present invention providesthat L₁ and L₄ independently are carboxy, thiocarboxy, methylamino,methylhydroxy, methylthio, ether or thioether.

The invention is also directed to methods for the preparation ofoligonucleosides with modified inter-sugar linkages. These modificationsmay be effected using solid supports which may be manually manipulatedor used in conjunction with a DNA synthesizer using methodology commonlyknown to those skilled in DNA synthesizer arts. Generally, the procedureinvolves functionalizing the sugar moieties of two nucleosides whichwill be adjacent to one another in the selected sequence. In a 5′ to 3′sense, the “upstream” nucleoside is generally modified at the 3′ sugarsite and is referred to hereinafter as “synthon 1”. In one process ofthe invention ribo- and 2′-deoxyribonucleosides of adenine, guanine,cytosine, uracil, thymine and their analogues are modified to give their3′-deoxy-3-hydroxymethyl analogues. These 3′-hydroxymethyl groups arethen converted into various types of electrophilic centers. This may beaccomplished in a number of ways such as the following, preferredscheme.

One class of starting materials, 3′-deoxy-3′-hydroxymethylribonucleosides, can be prepared as described by Townsend et al.,Tetrahedron Letters, 31:3101-3104 (1990), Samano, V. and M. J. Morris,Journal of Organic Chemistry, 55:5186-5188 (1990) and Bergstrom, D. E.,Nucleosides and Nucleotides 8(8): 1529-1535 (1989). Appropriate, known,selective sugar hydroxyl protection of these nucleosides followed bystandard 2′-deoxygenation procedures will afford the2′,3′-dideoxy-3′-hydroxymethyl-ribonucleosides. Nucleosides of this typecan be selectively protected and the 3′-hydroxymethyl moietyfunctionalized to a variety of suitable electrophilic moieties. Inaccordance with preferred embodiments of this invention, suchelectrophilic moieties include halomethyl, trifluoromethyl,sulfonylmethyl, p-methylbenzene sulfonylmethyl, hydrazinomethyl or3′-C-formyl.

The “downstream” nucleoside is generally modified at the 5′ sugar siteand is referred to hereinafter as “synthon 2”. Modification to produceribo and 2′-deoxyribonucleosides of adenine, guanine, cytosine, uracil,thymine and their analogues, with their 5′-hydroxymethylene groupconverted into various types of electrophilic centers can beaccomplished through various procedures using commercially availablenucleosides. For example, 5′-deoxy-5′-halo nucleoside, 5′-deoxy-5′-tosylnucleosides, and 5′-aldehydic nucleosides have been prepared by Jones,G. H. and J. G. Moffatt in Journal of the American Chemical Society90:5337-5338 (1968).

In general, synthon 1 may be represented as comprising the structure:

while synthon 2 generally comprises the structure:

wherein B_(x) is a variable base moiety; Q is O, CH₂, CHF or CF₂; and E₁and E₂ are the same or different and are electrophilic reactive groups.

The two synthons are coupled via a linking group reactive with theelectrophilic reactive groups or otherwise. Coupling between synthon 1and synthon 2 may occur either stepwise or in a concerted manner and mayresult in dinucleosides linked through the modified linkage of thepresent invention or may result in a chain of nucleosides, each of whichmay be linked to the next through said modified linkage.

Coupling via a concerted action may occur between the electrophiliccenters of synthon 1 and synthon 2 such as in the presence of ammonia oran ammonia derivative to produce a dinucleoside. A preferred embodimentof the present invention is the coupling of known, bromomethyl typesynthons by the addition of hydrazine to produce a preferred linkagehaving —L₁—L₂—L₃—L₄— equal to —CH₂NHNHCH₂—. Another preferred embodimentof the present invention is the coupling of bromomethyl type synthons bythe addition of hydroxylamine to produce a linkage having —L₁—L₂—L₃—L₄—equal to —CH₂NHOCH₂— or —CH₂ONHCH₂—.

Another procedure whereby inter-sugar linkages may be modified toprovide the dinucleoside structure described herein is via a Wittigreaction. Preferably, the starting material of such reaction is a3′-keto nucleoside such as described by Townsend, et al. in TetrahedronLetters 31:3101-3104 (1990); Samano, V. and M. J. Morris in Journal ofOrganic Chemistry 55:5186-5188 (1990); and Bergstrom, D. E., et al. inNucleosides and Nucleotides 8(8):1529-1535 (1989); or a 5′-aldehydicnucleoside as described by Jones, G. H. and J. G. Moffatt in Journal ofthe American Chemical Society 90:5337-5338 (1968). The starting materialis preferably reacted with a phosphorus ylide having a benzyl or otherprotecting group. One preferred ylide useful for this invention istriphenylphosphorane-benzyloxymethylidine. Another useful ylidepreferably used for this invention istriphenylphosphorane-benzyloxyethylidine. Reduction of the vinyl groupand hydrogenolysis of the benzyl protecting group provides hydroxymethyland hydroxyethyl moieties respectively, in the 5′ or 3′ positions of thedesired nucleoside of guanine, adenine, cytosine, thymine, uracil or theanalogues of these nucleosides. In addition, the Wittig reaction may beused to provide the 5′ and 3′ hydroxy alkyl moieties of carbocyclicnucleosides.

Conversion of the hydroxyl groups to provide electrophilic centers andsubsequent coupling of a 3′ electrophilic center with a 5′ electrophiliccenter will afford dinucleosides of the present invention. In oneembodiment of the invention, the hydroxyl groups are converted toprovide electrophilic centers such as bromides, triflates, andtosylates. Coupling affords dinucleosides connected by a carbon chainwith one or two heteroatoms. Preferably such heteroatoms may be O, NH,NR₃, S, SO, SO₂, P(O)R₄, P(S)R₄ or SiR₅R₆ as depicted in the genericformula provided previously.

Other useful dinucleosides which likely may be derived from a Wittigreaction involving 3′ or 5′ carbonyl nucleosides andtriphenylphosphorine methylidine diphenylphosphonate are phosphonatedinucleosides. This reaction provides the methyl or ethyl phosphonatewhich can be condensed with the corresponding 5′- or 3′-hydroxy group toprovide 3′- or 5′-phosphonate linked oligonucleosides. Chemistry of thistype has been described in the preparation of phosphonates ofdinucleosides for the study of biochemical processes, Moffatt, J. G., etal., Journal of American Chemical Society 92:5510-5513 (1970) and Mazur,A., B .E. Tropp, and R. Engel, Tetrahedron 40:3949-3956 (1984).Utilizing this type of coupling a preferred embodiment is prepared bythe coupling a 3′-keto nucleoside to a 5′-nucleoside with a symmetricalbis(methyltriphenylphosphane)phenylphosphate to provide3′,5′-dimethylphosphonate linked oligonucleotides.

In addition to the Wittig reaction, 3′-hydroxymethyl nucleosides mayalso be prepared through the inversion of alpha carbocyclic nucleosides.This will provide the desired 3′ hydroxymethyl group on the “down” oralpha face. This group can now be protected and the 3″-hydroxyl group(identifying the exo-cyclic methyl linked to the sugar 3′ position as 3″methyl) can be converted to an hydroxymethyl or longer alkyl group. Onemethod of converting the 3″ group involves oxidation to the keto groupfollowed by a Wittig reaction with triphenylphosphorine methylidinediphenylphosphonate and reduction. Longer hydroxyalkyl groups can beplaced in the 3″-position in this manner. This embodiment also providesa 4′-desmethyl-3′-hydroxymethyl nucleoside synthon. Coupling betweenthis 4′-desmethyl and the normal 3′-hydroxy-nucleoside with a two atomcoupler will provide dinucleoside synthons as described in prior pendingapplication (Ser. No. 566,836 filed Aug. 13, 1990, now U.S. Pat. No.5,223,618, which is assigned to the assignee of this application).Coupling of the 4′-desmethyl hydroxyl group with appropriate 3′-synthonsas described above will provide a number of other types of noveldinucleoside synthons.

Yet another approach to functionalize the methyl group of3′-deoxy-3′-methyl nucleosides may be elaborated from3′-deoxy-3′-cyanonucleosides. Parkes, K. E. B., and K. Taylor,Tetrahedron Letters 29:2995-2996 (1988) described a general method ofsynthesis of 3′-cyano nucleosides. In this method, 5′-trityl protected2′-deoxynucleosides are 3′-iodinated with methyltriphenylphosphoniumiodide. These materials are then treated with hexamethylditin,t-butylisonitrile, and 2,2′-azo-bisisobutrylonitrile (AIBN) to providethe radical addition of a cyano group to the 3′-position. Conversion ofthe cyano group to the aldehyde was accomplished in high yield.Subsequently, the intermediate was converted to hydroxymethyl functionswhich are valuable precursors to the electrophilic synthon 1.

An additional procedure whereby inter-sugar linkages may be modified toprovide dinucleosides utilizes 3′-C-formyl derivatized nucleosides assynthon 1 and 5′-aminohydroxy derivatized nucleosides as synthon 2.Direct coupling of synthons 1 and 2 gave a dinucleoside coupled via anoxime linkage. In this instance the oxime is present as E/Z isomers. Theisomeric compounds are separated utilizing HPLC. Further in thisinstance the oxime nitrogen atom is adjacent to a carbon atom on the 3′end of the upstream nucleoside. Dinucleosides having the oxime nitrogenadjacent to a carbon atom on the 5′ or downstream nucleoside aresynthesized utilizing a 5′-C-formyl derivatized nucleoside as synthon 2and a 3′-deoxy-3′-aminohydroxymethyl derivatized nucleoside assynthon 1. In this instance oxime E/Z isomers are also obtained. In bothinstances the oxime linked dimers are useful for direct incorporationinto an oligomer or then can be reduced to the correspondinghydroxyamino linked dinucleoside. Reduction of oxime linkeddinucleosides either as the dinucleoside or as a dinucleoside moiety inan oligomer with sodium cyanoborohydride yields the correspondingaminohydroxyl linked compounds. The hydroxyamino linked dinucleoside ora large oligomer could be alkylated at the amino moiety of theaminohydroxyl linkage to yield a corresponding N-alkylamino linkage.

The 3′-C-formyl derivatized synthon 1 can be formed via severalsynthetic pathways. The presently preferred method utilizes a radicalcarbonylation of the corresponding 3′-deoxy-3′-iodo nucleoside. The iodocompound is treated with CO, AIBN, i.e. 2,2′-azobisisobutrylnitrile, andTTMS, i.e. tris(trimethylsilyl)silane. Alternately it can be synthesizedfrom either a 3′-deoxy-3′cyano sugar or nucleoside. Both 5′-C-formyl(also identified as 5′-aldehydo) and 3′-C-formyl group can be blocked ina facile manner utilizing o-methylaminobenzenthiol as a blocking group.Both of the 5′ and the 3′-C-formyl groups can be deblocked with silvernitrate oxidation.

In an alternate method of 3′-C-formyl nucleoside synthesis,1-O-methyl-3′-deoxy-3′-O-methylaminobenzenethiol-5′-O-trityl-β-D-erythro-pento furanoside can be used for itspreparation. This compound then serves as a precursor for any3′-deoxy-3′-C-formyl nucleoside. The 1-O-methyl-3′-deoxy-3′-O-methylamino benzenethiol-5′-O-trityl-β-D-erythro-pentofuranoside is reactedwith an appropriate base utilizing standard glycosylation conditionsfollowed by deblocking to yield the nucleoside. In even a furtheralternate method a 3′-deoxy-3′-cyano nucleoside is prepared from eitherthe corresponding 3′-deoxy-3′-iodo nucleoside or via a glycosylationreaction with1-O-methyl-3′-deoxy-3′-O-cyano-5′-O-trityl-β-D-erythro-pentofuranoside.

The 3″-O-amino-3″-hydroxymethyl nucleoside and the corresponding5′-O-amino nucleoside can be conveniently prepared via a protectedphthalimido intermediate via Mitsunobu conditions usingN-hydroxyphthalimide, triphenylphosphine anddiisopropylazodicarboxylate. This in turn is prepared by a Mitsunobureaction on the unprotected hydroxyl group of the nucleoside. In formingthe 3″-O-amino-3″-hydroxymethyl nucleoside, trityl serves as a blockinggroup for the 5′-hydroxyl group of the nucleoside. For both purine andpyrimidine nucleosides prior to reacting with N-hydroxyphthalimide the3′-hydroxy group is protected with TBDPS. With pyrimidine bases, informing the 5′-O-amino nucleoside the 3′-hydroxyl can be protected withTBDPS blocking groups after introduction of the phthalimido on the 5′position.

A further procedure whereby inter-sugar linkages may be modified toprovide phosphonate linked dinucleotides utilizes the Michaelis-Arbuzovprocedure of Mazur et al., Tetrahedron, 20:3949 (1984) for formation of3′-C-phosphonate dimers. This procedure would utilize a 3′-hydroxymethylnucleosides as synthon 1. This is treated with N-bromosuccinimide toyield the corresponding 3″-bromomethyl derivative. Synthon 2 is selectedas a 5′-phosphite. Coupling of synthons 1 and 2 gives a dinucleosidecoupled via a 3′-C-phosphonate linkage. The corresponding5′-C-phosphonate dimers could be obtained by first reacting a suitableblocked phosphite with synthon 1 followed by deblocking to yield the3′-CH₂-phosphite intermediate. Synthon 2 is selected as a5′-bromonucleoside. The 3′-CH₂-phosphite intermediate is then reactedwith synthon 2 to give the 5′-C-phosphate dimer. By selectingtribenzylphosphite as the blocked phosphite after coupling to synthon 1the benzyl groups can be removed by hydrogenolysis. Alternately a5′-deoxy-5′-bromonucleoside is reacted with a phosphite ester resultingin a 5′-phosphonate. This in turn is reacted with 3′-hydroxymethylnucleoside to yield the 5′-C-phosphonate linked dimer.

Resulting dinucleosides from any of the above described methods, linkedby hydrazines, hydroxyl amines and other linking groups of theinventions, can be protected by a dimethoxytrityl group at the5′-hydroxyl and activated for coupling at the 3′-hydroxyl withcyanoethyldiisopropylphosphite moieties. These dimers may be insertedinto any desired sequence by standard, solid phase, automated DNAsynthesis utilizing phosphoramidite coupling chemistries. Therefore, theprotected dinucleosides are linked with the units of a specified DNAsequence utilizing normal phosphodiester bonds. The resultingoligonucleotide analogue or oligomer has a mixed backbone—part normalphosphodiester links and part novel four atoms links of the inventions.In this manner, a 15-mer, sequence-specific oligonucleotide can easilybe synthesized to have seven hydroxylamine, hydrazine or other typelinked dinucleosides. Such a structure will provide increased solubilityin water compared to native phosphodiester linked oligonucleotides.

Oligonucleosides containing an uniform backbone linkage can besynthesized by use of CPG-solid support and standard nucleic acidsynthesizing machines, i.e., Applied Biosystems Inc. 380B and 394 andMilligen/Biosearch 7500 and 8800 s. The initial nucleoside (number 1 atthe 3′-terminus) is attached to a solid support such as controlled poreglass and in sequence specific order each new nucleoside is attachedeither by manual manipulation or by the automated synthesizer system. Inthe case of a methylenehydrazine linkage, the repeating nucleoside unitcan be of two general types, e.g., a nucleoside with a 5′-protectedaldehydic function and a 3′-deoxy-3′-C-hydrazinomethyl group, or anucleoside bearing a 5′-deoxy-5′-hydrazino group protected by an acidlabile group and a 3′-deoxy-3′-C-formyl group. In each case, theconditions which are repeated for each cycle to add the subsequentsequence required base include: acid washing to remove the 5′-aldehydoprotecting group; addition of the next nucleoside with a3′-methylenehydrazino group to form the respective hydrazone connection;and reduction with any of a variety of agents to afford the desiredmethylene-hydrazine linked CPG-bound oligonucleosides. One such usefulreducing agent is sodium cyanoborohydride.

A preferred method is depicted in FIG. 1. This method employs a solidsupport on which a synthon 2 with a protected 5′ site is bound.Preferably, the 5′ site of said synthon may be protected with DMT.Thereafter, the 5′ site of the synthon 2 is liberated with mild acid,washed, and oxidized to produce an intermediate product. In onepreferred method, the aldehyde derivative reacts withN,N-diphenylethylene diamine to produce an intermediary product,5′-diphenylimidazolidino protected synthon 2. In a more preferred methodthe 5′-diphenylimidazolidino protected synthon 2 is directly loaded onthe support. With either method the intermediary product may besubsequently deblocked to provide a synthon 2 with a nucleophilic 5′position. Addition of a synthon 1 with a protected 5′-aldehyde group,such as a 5′-diphenylimidazolidino protected 3′-deoxy-3′-C-hydrazinebase, may then react, such as by the addition of sodiumcyanoborohydride, with the attached synthon 2. Following a wash, adinucleoside linked through a hydrazino moiety is formed. Thereafter,the cycle may be repeated as desired by the addition of a synthon 1species followed by acid/base deprotection to create a polysynthon, aresulting oligomer, of a desired sequence, linked together throughmodified inter-sugar linkages. In some preferred embodiments of thisinvention, the synthon 1 species may be a 5′-DMT protected3′-C-hydrazine base.

One preferred embodiment of this stepwise process utilizes adiphenylethyldiamine adduct (1,3-disubstituted imidazolidino) to protectthe electrophilic center of synthon 2 during attachment to the solidsupport. Moffatt, J. G., et al., Journal of American Chemical Society90:5337-5338 (1968). Synthon 2 may preferably be attached to a solidsupport such as a controlled pore glass support or other suitablesupports known to those skilled in the art. Attachment may take placevia a standard procedure. Gait, M. J., ed., Oligonucleotide Synthesis, APractical Approach (IRL Press 1984). Alternatively, preparation mayoccur by directly oxidizing the protected bound nucleoside with variousstandard oxidizing procedures. Bound synthon 2 is preferably reactedwith hydrazine to produce a Schiff's base which may be subsequentlyreduced. Hydroxyamine is also a preferred reactant useful in thismethod.

A further method of synthesizing uniform backbone linkedoligonucleosides is depicted in FIG. 2. This method also employs a solidsupport on which a synthon 2, with a protected 5′ site is bound. In thisinstance the 5′ site of the synthon is protected with a phthalimidogroup. Thereafter, the 5′ site of the synthon 2 is liberated withmethylhydrazine in DCM and washed with DCM:methanol. The aminohydroxylgroup at the 5′ position of synthon 1 is also protected with aphthalimido group. Such synthon 1 is a 5′-phthalimido protected3′-deoxy-3′-C-formyl nucleoside. Synthon 1 is reacted with synthon 2followed by deprotection at the 5′ position and washing to liberate thenext 5′-aminohydroxy reaction site. The cycle is repeated with thefurther addition of synthon 1 sufficient times until the desiredsequence is constructed. Each nucleoside of this sequence is linkedtogether with an oxime linkage. The terminal nucleoside of the desiredoligonucleoside is added to the sequence as a 5′-DMT blocked3′-deoxy-3′-C-formyl nucleoside. The oxime linked oligonucleoside can beremoved from the support. If a aminohydroxyl linked oligonucleoside isdesired the oxime linkages are reduced with sodium cyanoborohydride.Alternately reduction can be accomplished while the oxime linkedoligonucleoside is still connected to the support.

Also in accordance with this invention, nucleosides are provided havingthe structure:

wherein B_(x) is a variable base moiety; Q is O, CH₂, CHF or CF₂; X isH; OH; C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl oraralkyl; F; Cl; Br; CN; CF₃; OCF₃; OCN; O—, S—, or N-alkyl; O—, S—, orN-alkenyl; SOCH₃; SO₂CH₃; ONO₂; NO₂; N₃; NH₂; heterocycloalkyl;heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl;an RNA cleaving group; a group for improving the pharmacokineticproperties of an oligonucleotide; or a group for improving thepharmacodynamic properties of an oligonucleotide.

In such species, Y is hydroxyl, aminomethyl, hydrazinomethyl,hydroxymethyl, C-formyl, phthalimidohydroxymethyl, aryl-substitutedimidazolidino, aminohydroxylmethyl, methylaminobenzenethio,methylphosphonate and methyl-alkyl phosphonate; and Z is H, hydroxyl,aminomethyl, hydrazinomethyl, hydroxymethyl, C-formyl,phthalimidohydroxymethyl, aryl substituted imidazolidino,aminohydroxylmethyl, ortho-methylaminobenzenethio, methylphosphonate ormethyl alkylphosphonate.

All of the foregoing is with the proviso that when Q is O and Y ishydroxymethyl and X is H or OH then Z is not H or C-formyl; and when Qis O and X is H or OH and Z is hydroxyl then Y is notaminohydroxylmethyl, hydrazinomethyl or aryl-substituted imidazolidino.

The oligonucleotide analogues of this invention can be used indiagnostics, therapeutics, and as research reagents and kits. Fortherapeutic use the oligonucleotide analogue is administered to ananimal suffering from a disease modulated by some protein. It ispreferred to administer to patients suspected of suffering from such adisease an amount of oligonucleotide analogue that is effective toreduce the symptomology of that disease. One skilled in the art maydetermine optimum dosages and treatment schedules for such treatmentregimens.

It is generally preferred to administer the therapeutic agents inaccordance with this invention internally such as orally, intravenously,or intramuscularly. Other forms of administration, such astransdermally, topically, or intra-lesionally may also be useful.Inclusion in suppositories may also be useful. Use of pharmacologicallyacceptable carriers is also preferred for some embodiments.

EXAMPLES

The following examples are illustrative, but not limiting, of theinvention. In these examples, for the NMR of dimers and other higheroligonucleosides, the monomeric units of the dimer and other higheroligonucleosides are numbered, i.e T₁, T₂, from the 5′ terminusnucleoside towards the 3′ terminus nucleoside—thus the 5′ nucleoside ofa T-T dimer is T₁ and the 3′ nucleoside is T₂.

Example 1 Synthesis of CPG-bound Nucleosides for methylenehydrazine,i.e. (3′-CR₂—NH—NH—CH₂-5′), Linked Oligonucleoside

Diphenylimidazolidino Protected 5′-aldehydic thymidine

CPG-bound thymidine (30 micromoles of thymidine on one gram of CPGsupport, ABI, Foster City, Calif.) is treated at ambient temperaturewith a mixture of DMSO, benzene, DCC, pyridine, and trifluoroacetic acid(15 ml/15 ml/2.48 g/0.4 ml/0.2 ml in a procedure similar to theoxidation procedure of Pfitzer, K. E. and J. G. Moffatt, Journal ofAmerican Chemical Society 85:3027 (1963), to provide the 5′-aldehydicnucleoside. The mixture is filtered after storing overnight. The supportis washed with oxalic acid (1.3 g in 5 ml benzene/DMSO, 1 to 1) andtreated with 1,2-dianilinoethylene (3.0 g) for one hour, filtered, andwashed with acetonitrile to afford the 5′-diphenylimidazolidinoprotected 5′-aldehydic thymidine.

5′-Deoxy-5′-hydrazino-thymidine

Treatment of the support-bound 5′-aldehydo thymidine with a solution ofhydrazine hydrate/sodium cyanoborohydride in acetonitrile providesCPG-3′-bound 5′-deoxy-5′-hydrazino thymidine which is stored as itshydrochloride salt.

5′-Diphenylimidazolidino Protected-3′-deoxy-3′-C-hydrazinomethylthymidine

Commercially available 3′-O-acetylthymidine was oxidized andsubsequently protected as its N,N-diphenylethylenediamine derivative(1,3-diphenylimidazolidino). This provides the known5′-deoxy-5′-diphenylimidazolidino-3′-O-acetylthymidine. Pfitzer, K. E.and J. G. Moffatt, Journal of American Chemical Society 85:3027 (1963).Hydrolysis of this material was achieved by methanolic ammonia treatmentat ambient temperature for 15 hours.5′-Deoxy-5′-diphenylimidazolidinothymidine (4.5 g) was dissolved in DMF(100 ml) and treated with triphenylmethyl phosphonium iodide at roomtemperature for 15 hours. The solvent was removed under reduced pressureand the resulting residue recrystallized from methanol to provide the3′-deoxy-3′-iodo derivative. The 3′-deoxy-3′-iodo-5′-diphenylimidazolinothymidine was dissolved in toluene and treated with hexamethylditin,t-butylisonitrile, and AIBN. This radical reaction provides the3′-deoxy-3′-cyano derivative which was subsequently reduced withdiisobutylaluminum hydride (DIBAL-H) in toluene/THF at 0° C., to afford3′-deoxy-3′-C-formyl-5′-diphenylimidazolidino thymidine. This materialwas treated with hydrazine hydrate and sodium cyanoborohydride inacetonitrile to afford 5′-diphenylimidazolidinoprotected-3′-deoxy-3′-C-hydrazinomethyl thymidine. The material isconveniently stored as the acetate salt.

Example 2 Synthesis of Uniform (3′-CH₂—NH—NH—CH₂-5′), i.e.methylenehydrazine, Linked Oligonucleosides on a DNA Synthesizer

CPG-bound thymidine with a diphenylimidazolidino protected 5′-aldehydefrom Example 1 that will become the 3′-terminal nucleoside is placed inan Applied Biosystems, Inc. (ABI) column (250 mg, 10 micromoles of boundnucleoside) and attached to an ABI 380B automated DNA Synthesizer. Theautomated (computer controlled) steps of a cycle that are required tocouple a desmethyl nucleoside unit to the growing chain are as follows.

STEP REAGENT OR SOLVENT MIXTURE TIME (min:sec) 1 3% DCA indichloroethane 3:00 2 Dichloroethane wash 1:30 35′-Deoxy-5′-(1,3-diphenylimidazolidino)- 3′-deoxy-3′-C-methylenehydrazine nucleoside (the second nucleoside); 20 micromoles in 30 ml ofacetonitrile 2:50 4 Sodium borohydride (50 micromole in 1:1  THF/EtOH,50 ml) 3:00 5 Dichloroethane wash 2:00 6 Recycle starting at step 1(acid wash) 3:00This procedure yields as its product nucleoside the 5′-dimethyoxytritylsubstituted nucleoside unit.

At the completion of the synthesis, base deprotection and oligomerremoval from the support is accomplished by the standard proceduredescribed in oligonucleotide Synthesis: a practical approach, Ed. M. J.Gait, IRL Press, 1984. Trityl-on HPLC purification followed by aceticacid deprotection and precipitation provides the oligonucleosides as theacetate salts.

Example 3 Synthesis of 5′-deoxy-5′-hydrazino Nucleosides

5′-Deoxy-5′-hydrazinothymidine hydrochloride

To provide 5′-benzylcarbazyl-5′-deoxythymidine, 5′-O-tosylthymidine,[Nucleosides & Nucleotides 9:89 (1990)] (1.98 g, 5 mmol),benzylcarbazide (4.15 g, 25 mmol), activated molecular sieves (3A, 2 g),and anhydrous dimethylacetamide (100 ml) were stirred together withexclusion of moisture at 110° C. (bath temperature) for 16 hours. Theproducts were cooled and concentrated under reduced pressure (bathtemperature <50° C.). The residue was purified on a silica gel column(5×45 cm) with CH₂Cl₂/MeOH (9:1, v/v) as the solvent. The homogeneousfractions were pooled, evaporated to dryness and the foam recrystallizedfrom EtOH to yield 0.7 g (36%) of 5′-benzylcarbazyl-5′-deoxythymidine;mp 201° C.; ¹H NMR (Me₂SO-d₆) δ 1.79 (s, 3, CH ₃), 2.00-2.18 (m, 2,C₂,CH ₂), 2.95 (t, 2, C₅,CH ₂), 3.75 (m, 1, C₄,H), 4.18 (m, 1, C₃,H),4.7 (brs, 1, O′₂NH), 5.03 (s, 2, PhCH ₂), 5.2 (d, 1, C₃,H), 6.16 (t, 1,C₁,H), 7.2-7.4 (m, 5, C₆ H ₅), 7.6 (s, 1, C₆ H), 8.7 (brs, 1, CH₂NH),11.2 (brs, 1, C₃NH).

To provide the hydrochloride salt of 5-′-deoxy-5′-hydrazinothymidine asa hygroscopic powder, a mixture of the above carbazate (0.78 g, 2 mmol)and palladium on charcoal (10%, 150 mg) in anhydrous MeOH/HCl (30 ml,2%, HCl by weight) was stirred under an atmosphere of hydrogen at roomtemperature for 1.5 hours. The methanolic solution was filtered throughCelite to remove the catalyst. The filter cake was washed with EtOH(2×25 ml). The filtrate was concentrated under vacuum and the residuewas dried overnight to remove traces of HCl. The yellow residue wasdissolved in methanol (3 ml) and added dropwise to a rapidly stirredsolution of ethyl acetate (150 ml). The filtered precipitate was washedwith ethyl acetate (3×100 ml) and the pale yellow solid was dried undervacuum to yield 0.51 g (88%) of 5′-deoxy-5′-hydrazinothymidinehydrochloride (hygroscopic powder); ¹H NMR (Me₂SO-d₆) δ 1.81 (s, 3, CH₃), 2.02-2.22 (m, 2, C₂,CH ₂), 3.2 (m, 2, C₅,CH ₂), 3.8, (m, 1, C₄,H),4.2 (m, 1, C₃,H), 6.17 (t, 1, C₁, H), 7.54 (s, 1, C₆ H), 11.18 (brs, 1,C₃NH), the hydrazino and 3′-OH were masked by H₂O.

Example 4 Synthesis of5′-trityl-1-[2,3-dideoxy-3-C-(formyl)-β-D-erythro-pentofuranosyl]-thymineand -uracil

Method A

3′-C-Cyano-3′-deoxy-5′-O-tritylthymidine

“The following preparation should to be performed under a hood and allprecautions taken not to inhale any of reagent fumes.”

A suspension of 3′-deoxy-3′-iodo-5′-O-tritylthymidine (Verheyden, J. P.H.; Moffatt, J. G., J. Org. Chem., 35:2868 (1970)) (60 g, 0.1 mol),hexamethylditin (36 g, 22.7 ml, 0.11 mol), t-butylisocyanide (166 g, 225ml, 2 mol), and AIBN (1.6 g, 10 mmol) in toluene (freshly distilled overNa/benzophenone, 2 lt) was thoroughly deoxygenated by bubbling argonthrough the reaction mixture for 30 min. and then heated at 38° C. for13 h. The reaction mixture was cooled at 60° C. and AIBN (1.6 g, 10mmol) was added and heating continued for 24 h. During this periodaddition of AIBN was repeated for 3 times in an identical manner. Thereaction mixture was cooled to room temperature and transferred on thetop of a prepacked silica gel column (1.5 kg, in hexanes) and elutedwith hexanes: Et₂O (100% hexanes→100% Et₂O with a 10% gradient changeeach time using 1 lt of eluent). Most of the impurities were removedduring the gradient elution as non-polar compounds. Final elution withEt₂O (2 lt), pooling and evaporation of appropriate fractions gave twocompounds in the order these were collected. (i) 12.93 g (25%) of3′-C-Cyano-3′-deoxy-5′-O-tritylthymidine as white powder (crystallizedfrom toluene/Et₂O, mp 153-157° C.); ¹H NMR (CDCl₃) δ 8.83 (s, 1, NH),7.04-7.4 (m, 18.5, TrH, C₆ H, and 0.5 ArH from toluene), 6.10 (dd, 1, H_(1′), J_(1′,2′)=4.1 Hz, J_(1′,2″)=7.1 Hz), 4.20 (m, 1, H _(4′),J_(3′,4′)=8.4 Hz, J_(4′,5′)=2.8 Hz), 3.33-3.60(m, 3, H _(5′,5″,3′)) 2.68(m, 1, H _(2′), J_(2′,2″)=13.8 Hz), 2.52 (m, 1, H _(2″)), 2.28 (s, 1.5,0.5 CH ₃ from toluene), and 1.50 (s, 3, CH ₃). Anal. Calcd. forC₃₀H₂₇N₃O₄.0.5 C₇H₈ (toluene from crystallization): C, 74.56; H, 5.79;N, 7.78. Found: C, 74.27; H, 5.78; N, 7.66. The reaction mixture alsogave 4.82 g, (10%) of1-(3′-C-cyano-2′,3′-dideoxy-5′-O-trityl-β-D-threo-pentofuranosyl)-thymine;¹H NMR (CDCl₃) δ 8.72 (s, 1, NH), 7.03-7.44 (m, 18.5, TrH, C₆ H, and 0.5ArH from toluene), 6.13 (pseudo t, 1, H _(1′), J_(1′,2′)=6.7 Hz,J_(1′,2″)=5.7 Hz), 4.09 (m, 1, H _(4′), J_(3′,4′)=6.7 Hz, J_(4′,5′)=4.9Hz), 3.56 (m, 2, H _(5′,5″), 3.28 (m, 1, H _(3′), J_(3′,2′)=8.2 Hz,J_(3′,2″)=5.2 Hz), 2.70 (m, 1, H _(2′), J_(2′,2″)=14 Hz), 2.28 (s, 1.5,CH ₃ from toluene) and 1.60 (s, 3, CH₃). Anal. Calcd. for C₃₀H₂₇N₃O₄.0.5C₇H₈ (toluene from crystallization): C, 74.56; H, 5.79; N, 7.78. Found:C, 74.10; H, 5.74; N, 7.52.

Epimerization: To a suspension of1-(3′-C-Cyano-2′,3′-dideoxy-5′-O-trityl-β-D-threo-pentofuranosyl)thymine(0.30 g, 0.61 mmol) in methanol (20 ml) was added dropwise a 1N solutionof NaOMe until the pH of solution reached≈9. The resulting mixture washeated to reflux for 20 h. The solution was cooled (0° C.) andneutralized with 1N HCl/MeOH and evaporated under reduced pressure. Theresidue was purified as described above to furnish 0.185 g (62%) of3′-C-cyano-3′-deoxy-5′-O-tritylthymidine. (A synthesis for3′-deoxy-3′-C-cyano-5′-O-tritylthymine was reported in TetrahedronLetters 29:2995 (1988). This report suggested3′-deoxy-3′-C-cyano-5′-O-tritylthymine is formed as a single product,however, we found a mixture is produced. By the above epimerization, thexylo component of this mixture is converted to the compound of interest,3′-deoxy-3′-C-cyano-5′-O-tritylthymine.)

3′-Deoxy-3′-C-formyl-5′-O-tritylthymine

DIBAL-H (1M in toluene, 50 ml, in 5 portions over a period of 5 h) wasadded to a stirred solution of 3′-C-cyano-3′-deoxy-5′-O-tritylthymidine(9.92 g, 20 mmol) in dry THF (10 ml) under argon at 0° C. The solutionwas stirred at room temperature for 1 h and cooled again to 0° C. MeOH(25 ml) was added dropwise to the cold solution while stirring and aftercomplete addition the solution was brought to room temperature. Asaturated aqueous Na₂SO₄ solution (11 ml) was added to the reactionmixture and stirred for 12 h. Powdered anhydrous Na₂SO₄ (30 g) was addedto the reaction mixture and suspension was stirred for 30 min. Thesuspension was filtered and residue was thoroughly washed withMeOH:CH₂Cl₂ (1:9 v/v) until all of the product was washed off. Thefiltrates were combined and concentrated under vacuum, to furnish agummy residue. The residue was purified by silica gel chromatographyusing CH₂Cl₂:MeOH (100% CH₂Cl₂→9:1, v/v) for elution to obtain 5.45 g(55%) of 3′-deoxy-3′-C-formyl-5′-O-tritylthymine as a white foam. ¹H NMR(CDCl₃) δ 9.61 (d, 1, CHO, J_(3′,3″)=1.5 Hz), 8.44 (s, 1, NH), 7.46 (s,1, C₆ H), 7.17-7.45 (m, 15, TrH), 6.04 (pseudo t, 1, H _(1′),J_(1′,2′)=5.3 Hz, J_(1′,2″)=6.6 Hz), 4.31 (m, 1, H _(4′), J_(4′,5′)=3.3Hz, J_(3′,4′)=7 Hz), 3.28-3.55 (m, 3, H _(5′,5″,3′)), 2.69 (m, 1, H_(2′)), 2.28 (m, 1, H _(2″)), 1.48 (s, 3, CH₃). Anal. Calcd. forC₃₀H₂₈N₂O₅.H₂O: C, 70.03; H, 5.88; N, 5.44. Found: C, 70.40; H, 6.00; N,5.33.

1-[3-Deoxy-3-C-(formyl)-5-O-trityl-β-D-erythro-pentofuranosyl]uracil

To a stirred solution of 3′-cyano-2′,3′-dideoxy-5′-O-trityl uridine(0.96 g, 2 mmol), (prepared in a manner equivalent to that of thethymidine analogue above) in dry THF (20 ml) under argon, was added asolution of DIBAL-H in toluene (Aldrich) (1M, 4 ml) at −10° C. over aperiod of 10 min. After 30 mins the reaction was quenched with MeOH (5ml) at −10° C. The mixture was further stirred at ambient temperaturefor 30 mins and diluted with CH₂Cl₂ (25 ml) before concentrating undervacuum. This process was repeated with CH₂Cl₂ (3×25 ml) in order toremove the residual THF. The residue was purified by flashchromatography on silica gel (25 g). Elution with CH₂Cl₂ (9:1, v/v) andcrystallization from CH₂Cl₂/MeOH gave5′-O-trityl-3′-C-formyl-2′,3′-dideoxyuridine (0.53 g, 53%); mp 100° C.;¹H NMR (CDCl₃) δ 2.25-2.8 (m, 2, CH ₂), 3.4 (m, 1, C_(3′) H), 3.45-3.6(m, 2, C_(5′)CH ₂), 4.37 (m, 1, C_(4′) H), 5.4 (d, 1, C₅ H), 6.1 (m, 1,C_(1′) H), 7.2-7.4 (m, 15, C₆ H ₅), 7.81 (d, 1, C₆ H), 7.95 (br s, 1,NH), 9.61 (s, 1, HC═O).

Method B

1-[3-deoxy-3-C-(formyl)-5-O-trityl-β-D-erythro-pentofuranosyl]thymine

1-Methyl-5-O-(t-butyldiphenylsilyl)-2,3-dideoxy-3-C-(formyl)-D-erythro-pentofuranosewas obtained as an oil in 90% yield using the DIBAL-H reduction of1-methyl-5-(t-butyldiphenylsilyl)-2,3-dideoxy-3-C-cyano-D-erythro-pentofuranoseas described in Tetrahedron, 44:625 (1988). The 3-C-formyl group isderivatized to the oxime with methoxyamine. The oxime blockedintermediate was glycosylated with silyated thymine to give an a and 8mixture of the title compound. After deblocking, the 8 anomer comparesto that prepared via method A.

Method C

1-[3-deoxy-3-C-(formyl)-5-O-trityl-β-D-erythro-pentofuranosyl]-uraciland -thymine

A mixture of 3′-deoxy-3′-iodo-5′-O-tritylthymidine (0.59 g, 4 mmol),tris(trimethylsilyl) silane (2.87 g, 1.2 mmol), AIBN (12 mg, 0.072mmol), and toluene (20 ml) were mixed in a glass container and saturatedwith argon (bubbling at room temperature). The glass vessel was insertedinto a stainless steel pressure reactor, and pressurized with carbonmonoxide (80 psi), closed and heated (90° C., bath) for 26 hrs. Thereaction mixture was cooled (0° C.) and the CO was allowed to escapecarefully (under the fume hood). The product was purified by flashcolumn chromatography on silica gel (20 g). Elution with EtOAc:Hexanes(2:1, v/v) and pooling the appropriate fractions furnished 0.30 g (61%)of the title compound as a foam.

A radical carbonylation of 2′,3′-dideoxy-3′-iodo-5′-trityluridine in asimilar manner gives the 3′-C-formyl uridine derivative.

Example 5 Synthesis of methylenehydrazone Linked (3′-CH═NH—NH—CH₂-5′),methylenehydrazine Linked (3′-CN₂—NH—NH—CH₂-5′) andmethylene(dimethylhydrazo) Linked (3′-CH₂—N(CH₃)—N(CH₃)—CH₂-5′)dinucleosides

3′-De(oxyphosphinico)-3′-[methylene(hydrazone)]-5′-O-tritylthymidylyl-(3′→5′)-5′-deoxythymidine

A mixture of 3′-deoxy-3′-C-formyl-5′-O-tritylthymidine, 0.645 g, 1.30mmol), 5′-deoxy-5′-hydrazinothymidine hydrochloride (0.397 g, 1.36 mmol)in dry CH₂Cl₂/MeOH/AcOH (20 ml/10 ml/0.5 ml) was stirred for 30 min atroom temperature. The solvent was evaporated under vacuum and thehydrazone intermediate was analyzed by ¹H NMR (DMSO-d₆) δ 1.1 (br s, 2NH), 8.3 (s, 1, C═N—NH), 7.5-7.74 (m, 17, Tr H, 2C₆ H), 6.8 (1d, 1t, 1,HC═N, two isomers), 6.0-6.1 (2m, 2, H _(1′)), 5.30 (br t, 1, OH),3.8-4.2 (3m, 3, H _(3′), 2 H _(4′)), 3.0-3.3 (m, 5, 2H _(5′,5″), H_(3′)), 2.0-2.4 (m, 4, 2H _(2′,2)), 1.5 and 1.7 (2s, 6, 2 CH ₃).

3′-De(oxyphosphinico)-3′-[methylene(dimethylhydrazo)]-5′-O-tritylthymidylyl-(3′→5′)-5′-deoxythymidine

The above hydrazone dimer was dissolved in AcOH (10 ml) and to this wasadded small portions of NaBH₃CN (4×0.12 g, 7.74 mmol) while stirring atroom temperature for 30 min. The solution was stirred for an additional15 min before the addition of aqueous HCHO (20%, 3.9 ml, 26 mmol),NaBH₃CN (3.9 mmol), and AcOH (10 ml). The suspension was further stirredfor 15 min. and solution evaporated under vacuum. The residue wascoevaporated with MeOH (3×25 ml) to give the methylenehydrazo dimer; ¹HNMR (CDCl₃) δ 6.8-7.8 (m, 15, TrH, 2 C₆ H), 6.12 (m, 2, 2H _(1′)), 4.20((m, 1, T2 H _(3′)), 4.05 (m, 1, T2 H _(4′)), 3.89 (m, 1, T1 H _(4′)),3.80 (s, 6, 2 OCH ₃), 3.21-3.53 (m, 2, T1 H _(5′,5″)), 2.11-2.75 (m, 10,T2 H _(5′5″)H, T1 H _(3′), T1 H _(3′), T1 T2 H _(2′2″)) 2.26 (s, 6,2N—CH ₃), 1.88 and 1.49 (2s, 6, 2 CH ₃), and other protons.

3′-De(oxyphosphinico)-3′-[methylene(dimethylhydrazo)]-thymidylyl-(3′-5′)-5′-deoxythymidine

The above hydrazine dimer was then stirred with 37% aqueous HCl (1 ml)in MeOH (25 ml) at room temperature for 24 h. The resulting mixture wasneutralized with NH₄OH (pH≈8) and evaporated to dryness. The residue waspurified by reverse phase HPLC (supelcosil LC18, 5 m, H₂O: CH₃CNgradient) to furnish 0.61 g of the title methylene(dimethylhydrazine)linked dimer (89%). ¹H NMR (90° C., DMSO-d₆+1 drop of D₂O) δ 7.66 and7.43 (2s, 2, 2 C6H), 6.02 (pseudo t, 1, T2 H _(1′), J_(1′,2′)=7.2 Hz,J_(1′,2′)=7.7 Hz), 5.96 (pseudo t, 1, T1 H _(1′), J_(1′,2′)=5.6 H₂,J_(1′,2″)=6.2 Hz), 4.12 (m, 1, T2 H _(3′)), 3.90 (m, 1, T2 H _(4′)),3.71 (m, 1, T1 H _(4′)), 3.61 (m, 2, T1 H _(5′,5″)), 2.4-2.8 (m, 5, T2 H_(5′,5″), T1 H _(3″), T1 H _(3′)), 2.29 (2s, 6, 2 N—CH ₃), 2.12 (m, 4,2H_(2′,2″)), 1.76 and 1.74 (2s, 6, 2 CH ₃). Anal. Calcd. for C₂₃H₃₄N₆O₈,H₂O: C, 51.10, H, 6.71; N, 15.54. Found: C, 51.05; H, 6.68; N, 15.54. MSFAB m/z 523 (M+H)⁺.

Example 6 Synthesis of methylene(dimethylhydrazine) Linked(3′-CH₂—N(CH₃)—N(CH₃)—CH₂-5′)5′-dimethoxytrityl-3′-β-cyanoethoxydiisopropylphosphoramiditedinucleosides

3′-De(oxyphosphinico)-3′-[methylene(dimethylhydrazo)]-thymidylyl-5′-O-(dimethoxytriphenylmethyl)-(3′→5′)-3′-O-(β-cyanoethyl-N-diisopropylaminophosphiryl)thymidine

The methylene(dimethylhydrazine) dimer of Example 5 wasdimethyoxytritylated following the standard procedure described inoligonucleotide Synthesis: a practical approach, Ed. M. J. Gait, IRLPress, 1984, to furnish a homogenous foam ¹H NMR (CDCl₃) δ 6.8-7.8 (m,20, DMTr, 2H ₆), 6.12 (m, 2, 2H _(1′)), 4.20 (m, 1, T₂ H _(3′)), 4.05(m, 1, T₂ H _(4′)), 3.89 (m, 1, T₁ H _(4′)), 3.80 (s, 6, 2 OCH ₃ ofDMTr), 3.21-3.53 (m, 2, T₁ H _(5′5″)), 2.11-2.75 (m, 9, T₁ H _(5′5″), H_(3″), T₁ H_(3′), 2H _(2′2″)), 2.26 (2s, 6, 2 N—CH ₃) and 1.88 and 1.49(2s, 2, C₅ CH ₃₎] which on phosphitylation via the procedure describedin Oligonucleotide Synthesis: a practical approach, Ed. M. J. Gait, IRLPress, 1984, provided a 65% yield of the title compound. ¹H NMR (CDCl₃)δ 6.14 (m, 1, T2 H _(1′)), 6.01 (m, 1, T1 H _(1′)), 3.80 (s, 6, 2 O CH₃), 2.23 (m, 6, 2 N—CH ₃), 1.78 and 1.45 (2s, 6, 2 CH ₃), and otherprotons. ³¹P NMR (CDCl₃) δ 149.43 and 148.85 ppm.

Example 7 Synthesis of Intermittent methylene(dimethyhydrazine)(3′-CH₂—NCH₃—NCH₃—CH₂-5′) Linked Oligonucleosides

CPG-bound thymidine (or any other nucleoside that is to become the3′-terminal base) was placed in an Applied Biosystems, Inc. (ABI) column(250 mg, 10 micromoles of bound nucleoside) and attached to an ABI 3808automated DNA Synthesizer. The standard, automated (computer controlled)steps utilizing phosphoramidite chemistries are employed to place themethylenehydrazine thymidine dimer into the sequence at any desiredlocation.

Example 8 Synthesis of (3′-CH₂—NH—S—CH₂-5′) Linkage

3′-de(oxyphosphinico)-3′-[methylene(methylsulfenyl)]-thymidylyl-(3′→5′)-5′-deoxythymidine

The title compound will be prepared from two intermediate nucleosides.The first nucleoside, 3′-O-benzyl-5′-deoxy-5′-mercaptothymidine will beprepared in 3 steps from 3′-O-benzoylthymidine according to theprocedure of J. H. Marriott et al., Tet. Letts., 31:7385 (1990), via aformation of the 5′-S-[9-(4-methoxyphenyl)xanthen-9-yl] group andsubsequent deblocking to yield a 5′-SH group. The second nucleoside,3′-C-methylamino-5′-O-tritylthymidine will be prepared in 3 steps from3′-C-formyl-5′→O-tritylthymidine described in Example 4 above. The 3steps procedure includes NaBH₄ reduction of the formyl group followed byconversion to an azido group with LiN₃/DMF and subsequent reduction withTBTH/toluene to furnish the 3′-C—CH₂NH₂ group. Addition of3′-C-methylamino-5′-O-tritylthymidine nucleoside (1 mmol) to an aqueoussodium hypochloride (4 mmol) solution will furnish the chloramideintermediate, which on cooling (0 °C.) and reaction with the3′-O-benzyl-5′-deoxy-5′-mercaptothymidine nucleoside (0.9 mmol) for 15min followed by the usual work-up and purification by chromatographywill furnish the title compound.

Example 9 Synthesis of 5′-O-phthalimido Nucleosides

5′-O-Phthalimidothymidine

To a stirred solution of thymidine (24.22 g, 0.1 mol),N-hydroxyphthalimide (21.75 g, 0.13 mol), triphenylphosphine (34 g, 0.13mol) in dry DMF (400 ml) was added diisopropylazodicarboxylate (30 ml,0.15 mol) over a period of 3 h at 0° C. After complete addition thereaction mixture was warmed up to room temperature and stirred for 12 h.The solution was concentrated under vacuum (0.1 mm,<40° C.) to furnishan orange-red residue. The residual gum was washed several times withEt₂O and washing were discarded. The semi-solid residue was suspended inEtOH (500 ml) and heated (90° C.) to dissolve the product. On cooling30.98 g (80%) of 5′-O-phthalimidothymidine was collected in 3-crops aswhite crystalline material, mp 233-235° C. (decomp.); ¹H NMR (DMSO-d₆) δ11.29 (s, 1, NH), 7.85 (m , 4, ArH), 7.58 (s, 1, C₆ H), 6.20 (t, 1, H_(1′,2′)=7.8 Hz, J_(1′,2″)=6.5 Hz), 5.48 (d, 1, OH _(3′)), 4.36 (m, 3, H_(4′,5′,5″)), 4.08 (m, 1, H _(3′)), 2.09-2.13 (m, 2, H _(2′,2″)), and1.79 (s, 3, CH ₃). Anal. Calcd. for C₁₈H₁₇O₇N₃.0.7H₂O: C, 54.05; H,4.64; N, 10.51. Found: C, 53.81; H, 4.25; N, 10.39.

2′-deoxy-5′-O-phthalimidouridine

An analogous reaction on 2′-deoxyuridine gave the corresponding2′-deoxy-5′-O-phthalimidouridine; mp 241-242° C.

Example 10 Synthesis of5′-O-phthalimido-3′-O-(t-butyldiphenylsilyl)thymidine and2′-deoxy-5′-O-phthalimido-3′-O-(t-butyldiphenylsilyl)uridine

3′-O-(t-butyldiphenylsilyl)-5′-O-phthalimidothymidine

A mixture of 5′-O-phthalimidothymidine (8.54 g, 22 mmol),t-butyldiphenylsilylchloride (6.9 ml, 26.5 mmol), imidazole (3.9 g, 57.3mmol) and dry DMF (130 ml) was stirred at room temperature for 16 hunder argon. The reaction mixture was poured into ice-water (600 ml) andthe solution was extracted with CH₂Cl₂ (2×400 ml). The organic layer waswashed with water (2×250 ml) and dried (MgSO₄). The CH₂Cl₂ layer wasconcentrated to furnish a gummy residue which on purification by silicagel chromatography (eluted with EtOAc:Hexanes; 1:1, v/v) furnished 12.65g (92%) of 3′-O-(t-butyldiphenylsilyl)-5′-O-phthalimidothymidine ascrystalline material (mp 172-173.5° C.). ¹H NMR (DMSO-d₆) δ 11.31 (s, 1,NH), 7.83 (m, 4, ArH), 7.59 (m, 4, TBDPhH), 7.51 (s, 1, C₆ H), 7.37-7.45(m, 6, TBDPhH), 6.30 (dd, 1, H _(1′), J_(1′,2′)=8.8 Hz, J_(1′,2″)=5.6Hz), 4.55 (m, 1, H _(4′)), 4.15 (m, 1, H _(3′)) 3.94-4.04 (m, 2, H_(5′,5″)), 2.06-2.13 (m, 2, H _(2′,2″)), 1.97 (s, 3, CH ₃), 1.03 (s, 9,C(CH ₃)₃). Anal. Calcd. for C₃₄H₃₅O₇N₃Si: C, 65.26; H, 5.64; N, 6.71.Found: C, 65.00; H, 5.60; N, 6.42.

3′-O-(t-butyldiphenylsilyl)-2′-deoxy-5′-O-phthalimidouridine

An analogous reaction of 2′-deoxy-5′-O-phthalimidouridine will give thecorresponding3′-O-(t-butyldiphenylsilyl)-2′-deoxy-5′-O-phthalimidouridine.

Example 11 Synthesis of 5′-O-amino Nucleoside

5′-O-amino-3′-O-(t-butyldiphenylsilyl)thymidine

To a stirred solution of3′-O-(t-butyldiphenylsilyl)-5′-O-phthalimidothymidine (10 g, 16 mmol) indry CH₂Cl₂ (100 ml) was added methylhydrazine (1.3 ml, 24 mmol) underargon at room temperature and solution stirred for 12 h. The solutionwas cooled (0° C.) and filtered. The white residue was washed withCH₂Cl₂ (2×25 ml) and combined filtrates were evaporated to furnish gummyresidue. The residue on purification by silica gel column chromatography(elution with CH₂Cl₂:MeOH, 98:2, v/v) furnished 7.03 g (89%) of5′-O-amino-3′-O-(t-butyldiphenylsilyl)thymidine that crystallized fromCH₂Cl₂/MeOH mp 141-143° C. ¹H NMR (DMSO-d₆) δ 11.29 (s, 1, NH),7.42-7.62 (m, 11, TBDPhH, C₆ H), 6.25 (dd, 1, H _(1′), J_(1′,2′)=8.4 Hz,J_(1′,2′)=6.3 Hz), 6.02 (s, 2, NH ₂), 4.35 (m, 1, H _(4′)), 4.04 (m, 1,H _(3′)), 3.34-3.51 (m, 2, H _(5′,5″)) 2.04 (m, 2, H _(2′, 2″)), 1.73(s, 3, CH ₃), 1.03 (s, 9, C(CH ₃)₃). Anal. Calcd. for C₂₆H₃₃O₅N₃Si: C,63.00; H, 6.71; N, 8.48. Found: C, 62.85; H, 6.67; N, 8.32.

Example 12 Synthesis of (3′-CH═N—O—CH₂-5′) Linked Oligonucleoside (anOxime Linked Dimer)

3′-De(oxyphosphinico)-3′-(methylidynenitrilo)thymidylyl-(3′→5′)-thymidine

A mixture of 3′-deoxy-3′-C-formyl-5′-O-tritylthymine (0.99 g, 2 mmol),5,-amino-3-O-(t-butyldiphenylsilyl)thymidine (0.99 g, 2 mmol) and AcOH(0.3 ml) in dry CH₂Cl₂ (20 ml) was stirred for 1 h at room temperature.The solvent was evaporated under vacuum and the crude blocked3′-de(oxyphosphinico-3′-(methylidynenitrilo)thymidylyl-(3′→5′)-3′-(t-butyldiphenylsilyl)thymidineproduct was dissolved in THF (20 ml). A THF solution of nBU₄NF (1M, 5ml) was added to the stirred reaction mixture at room temperature. After1 h solution was purified by silica gel chromatography (elution withCH₂Cl₂:MeOH; 99:4, v/v) to furnish 3′-deblocked dimer. The dimer wasdissolved in anhydrous MeOH (50 ml) and to this a MeOH/HCl solution(0.14M, 2.5 ml) was added. The reaction mixture was stirred at roomtemperature for 15 h. Anhydrous pyridine (10 ml) was added to the abovesolution and solvents were evaporated to dryness to furnish crude oximedimer. The crude product was purified by silica gel chromatography(elution with CH₂Cl₂:MeOH; 92:8, v/v) to furnish the title compound,3′-De(oxyphosphinico)-3′-(methylidynenitrilo)thymidylyl-(3′→5′)-thymidine,(0.87 g, 89%) as a mixture of E/Z isomers. The two geometrical isomerswere separated by reverse phase HPLC (Supelcosil LC18, 5μ, H₂O:CH₃CNgradient). (Z-isomer of title compound) ¹H NMR (DMSO-d₆) δ 11.28 (br s,2, 2NH), 7.39 and 7.78 (2s, 2, 2C6H), 6.92 (d, 1, T1 H _(3″),J_(3′,3″)=6.7 Hz), 6.15 (pseudo t, 1, T2 H _(1′), J_(1′,2′)=7.8 Hz,J_(1′,2″)=6.3 Hz), 6.04 (dd, 1, T1 H _(1′), J_(1′,2′)=7.1 Hz,J_(1′,2″)=6.3 Hz), 5.34 (d, 1, T2 OH), 5.12 (t, 1, T1 OH), 4.11-4.25 (m,3, T2 H _(5′5″), T2 H _(3′)). 3.96 (m, 1, T2 H _(4′)), 3.90 (m, 1, T1 H_(4′)), 3.49-3.69 (m, 3, T1 H _(5′,5″), T1 H _(3′)), 2.06-2.31 (m,4, T1H _(2′,2″), T2 H _(2′,2″)), 1.73 (s, 6, 2CH ₃). Anal. Calcd. forC₂₁H₂₇N₅O₉.H₂O: C, 49.31; H, 5.72; N, 13.69. Found: C, 49.32; 5.57; N,13.59. (E-isomer of the title compound) ¹H NMR (DMSO-d₆) δ 11.3 (2 br s,2, 2NH), 7.81 (s, 1, C₆ H), 7.52 (d, 1, T1 H _(3″), J_(3′, 3″)=6.7 Hz),7.45 (s, 1, C₆ H), 6.10 (pseudo t, 1, T2 H _(1′), J_(1′,2′)=7.6 Hz,J_(1′,2″)=6.4 Hz), 6.04 (dd, 1, T1 H _(1′), J_(1′,2′)=7.3 Hz,J_(1′,2″)=3.4 Hz), 5.36 (d, 1, T2 OH), 5.16 (t, 1, T1 OH), 4.07-4.22 (m,3, T2 H _(3′,5′,5″)), 3.91 (m, 2, T1 T2 H ₄), 3.50-3.73 (m, 2, T1 H_(5′,5″)), 3.12 (m, 1, T1 H _(3′)), 2.05-2.44 (m, 4, T1 T2 H _(2′,2″))and 1.76 (s, 6, 2CH ₃). MS FAB: M/z 494 (M+H)⁺.

Example 13 Synthesis of Phosphoramidate Containing (3′-CH═N—O—CH₂-5′)Linked Oligonucleoside

3′-De(oxyphosphinico)-3′-methylidynenitrilo)-5′-O-(di-methyoxytriphenylmethyl)-thymidylyl-(3′→5′)-3′-O-(β-cyanoethyldiisopropylaminophosphiryl)thymidine

The isomeric dimer of Example 12 was further dimethyoxytrityled at thehydroxyl group of the 5′ terminus nucleoside followed by conversion toits 3′-O-β-cyanoethyldiisopropylphosphoramidite derivative at thehydroxyl group at the 3′ terminus nucleoside of the dimer following theprocedure described in Oligonucleotide Synthesis: a practical approach,Ed. M. J. Gait, IRL Press, 1984, to yield the title compound. ¹H NMR(CDCl₃) δ 8.77 (br s, 2, 2NH), 7.68 (s, 0.77, T1 C₆ H E-isomer), 7.59(s, 0.23, T1 C₆ H E-isomer), 6.3 (ps t, 1, T2 CH _(1′)), 6.14 (m, 0.77,T1 CH _(1′)E-isomer), 6.08 (m, 0.23, T₁ CH _(1′) Z-isomer), 1.80 and1.50 (2S, 6, 2 CH ₃) and other protons. ³¹P NMR (CDCl₃) 150.77 and150.38 (Z-isomer); 150.57 and 150.38 (E-isomer).

The protected dimer can be conveniently stored and used for couplingutilizing an automated DNA synthesizer (ABI 380B) as and when requiredfor specific incorporation into oligomers of therapeutic value. Furtheras per further examples of the specification, the oxime linked dimer isreduced to a dimer bearing a corresponding hydroxylamine linkage andthis in turn can be alkylated to a hydroxylmethylamine or otherhydroxyalkylamine linkage.

Example 14 Synthesis of (3′-CH₂—NH—O—CH₂-5′) Linked Oligonucleoside

3′-De(oxyphosphinico)-3′-(methyleneimino)thymidylyl-(3′→5′)-thymidine

To a stirred solution of blocked dimer3′-de(oxyphosphinico)-3′-(methylidynenitrilo)thymidylyl-(3′→5′)-3′-O-(t-butyldiphenylsilyl)thymidine(0.49 g, 1 mmol) in glacial AcOH (5 ml) was added NaBH₃CN (0.19 g, 3mmol) in 3-portions under argon at room temperature. The suspension wasstirred for 1 h until bubbling of solution ceased. Additional NaBH₃CN(0.19 g, 3 mmol) was added in a similar manner and stirring continuedfor 1 h. The AcOH was removed under reduced pressure to furnish3′-de(oxyphosphinico)-3′-(methyleneimino)thymidylyl-(3′→5′)-3′-O-(t-butyldiphenylsilyl)thymidine.Deblocking of this dimer as described before using nBu₄NF/THF andHCl/MeOH furnished the title compound,3′-de(oxyphosphinico)-3′-(methyleneimino)-thymidylyl-(3′→5′)-thymidine,(0.44 g, 90%) as white powder. This dimer was further purified by HPLC(as described for the3′-de(oxyphosphinico)-3′-(methylidynenitrilo)thymidylyl-(3′→5′)-thymidinedimer of Example 12) to obtain an analytically pure sample. ¹H NMR(DMSO-d₆) δ 11.23 (br s, 2, 2NH), 7.83 and 7.49 (2s, 2, 2C₆ H), 6.82 (t,1, NHO), 6.14 (pseudo t, 1, T2 H _(1′), J_(1′,2′)=7.6 Hz, J_(1′,2″)=6.5Hz), 5.96 (dd, 1, T1 H _(1′), J_(1′,2′)=6.9 Hz, J_(1′,2″)=4.3 Hz), 5.28(s, 1, T2 OH), 5.08 (s, 1, T1 OH), 4.18 (m, 1, T2 H _(3′)), 3.89 (m, 1,T1 H _(4′)), 3.54-3.78 (m, 5, T1 T2 H _(5′,5″), T2 H _(4′)), 2.76-2.94(m, 2, T1 H _(3″)), 2.42 (m, 1, T1 H _(3′)), 2.0-2.17 (m, 4, T1, T2 H_(2′,2″)), 1.77 and 1.74 (2s, 6, 2 CH ₃). MS FAB: M/z 496 (M+H)⁺. Anal.Calcd. for C₂₁H₂₉N₅O₉.H₂O: C, 49.12; H, 6.09; N, 13.64. Found: C, 48.99;H, 5.96; N, 13.49.

Example 15 Synthesis of Methylated [3′-CH₂—N(CH₃)—O—CH₂-5′] LinkedOligonucleoside

3′-De(oxyphosphinico)-3′-[methylene(methylimino)]thymidylyl-(3′→5′)thymidine

To a stirred solution of3′-de(oxyphosphinico)-3′-(methyleneimino)thymidylyl-(3′→5′)-3′-O-(t-butyldiphenylsilyl)thymidinedimer (0.99 g, 1 mmol) in glacial AcOH (10 ml) was added aqueous HCHO(20%, 3 ml). The solution was stirred for 5 min. at room temperature andto this was added NaBH₃CN (0.19 g, 3 mmol) in 3-portions under argon atroom temperature. The addition of NaBH₃CN (0.19 g) was repeated oncemore and solution was further stirred for 1 h. The reaction mixture wasconcentrated to furnish crude3′-de(oxyphosphinico)-3′-[methylene(methylimino)]thymidylyl-(3′→5′)-3′-O-(t-butyldiphenylsilyl)thymidinedimer, which on deblocking (nBu₄NF/THF, HCl/MeOH) furnished the titlecompound,3′-de(oxyphosphinico)-3′-[methylene(methylimino)]thymidylyl-(3′→5′)thymidine, (0.44 g, 87%) as white solids. The3′-de(oxyphosphinico)-3′-[methylene-(methylimino)]thymidylyl-(3′→5′)thymidine dimer was further purified by preparative HPLC furnishing ananalytically pure sample. ¹H NMR (DMSO-d₆) δ 11.30 and 11.24 (2s, 2,2NH), 7.82 and 7.50 (2s, 2, 2C₆ H), 6.15 (pseudo t, 1, T2 H _(1′),J_(1′,2′)=6.3 Hz, J_(1′,2″)=7.3 Hz), 6.00 (pseudo t, 1, T1 H _(1′),J_(1′,2′)=4.2 Hz, J_(1′,2″)=6.1 Hz), 5.31 (m, 1, T2 OH), 5.08 (m, 1, T1,OH), 4.17 (m, 1, T2 H _(3′)), 3.88 (m, 1, T2 H _(4′)), 3.57-3.83 (m, 5,T1 T2, H _(5′,5″), T1 H _(4′)), 2.69 (m, 2, T1 H _(3″)), 2.57 (s, 3,N—CH ₃), 2.50 (m, 1, T1 H _(3′)), 2.05-2.14 (m, 4, T1 T2 H _(2′,2″)),1.79 and 1.76 (2s, 6, 2 CH ₃). MS FAB: M/z 510 (M+H)⁺. Anal. Calcd. forC₂₃H₃₁N₅O₉.H₂O: C, 50.09; H, 6.31; N, 13.28. Found: C, 50.05; H, 6.21,N, 13.08.

Example 16 Synthesis of Phosphoramidate Containing[3′-CH₂—N(CH₃)—O—C₂-5′] Linked Oligonucleoside

3′-De(oxyphosphinico)-3′-[methylene(methylimino)]-5′-O-(dimethoxytriphenylmethyl)thymidylyl-(3′→5′)-3′-O-(β-cyanoethyldiisopropylaminophosphiryl)thymidine

The 3′-de(oxyphosphinico)-3′-[methylene(methylimino)]-thymidylyl-(3′→5′)thymidine dimer of Example 15 was tritylated and phosphitylated asdescribed in Oligonucleotide Synthesis: a practical approach, Ed. M. J.Gait, IRL Press, 1984, in an overall yield of 82%. The protected dimerwas purified by silica gel column chromatography (CH₂Cl₂:MeOH:Et₃N;9:1:0.1, v/v) and homogenous fractions were pooled and evaporated tofurnish pure3′-de(oxyphosphinico)-3′-[methylene(methylimino)]-thymidylyl-5′-O-(dimethoxytriphenylmethyl)-(3′→5′)-3′-O-(β-cyanoethyldiisopropylaminophosphiryl)thymidineas a white foam (used as such for DNA synthesis). The product wasisolated as a mixture of diastereoisomer: ³¹P NMR (CDCl₃) δ 149.62 and149.11 ppm; ¹H NMR (CDCl₃) δ 6.22 (pseudo t, 1, T2 H _(1′),J_(1′,2′)=J_(1′,2″)=6.7 Hz), 6.16 (pseudo t, 1, T1 H _(1′),J_(1′,2″)=5.8 Hz), 2.58, 2.56 (2s, 3, N—CH ₃), 1.82, 1.49 (2s, 6, 2 CH₃), and other protons.

The above protected phosphoramidate bearing dimer can be convenientlystored and used for coupling utilizing an automated DNA synthesizer (ABI380B) as and when required for specific incorporation into oligomers oftherapeutic value. Other dimers of the inventions, as for example butnot limited the above noted methylidynenitrilo, i.e. oxime, andmethyleneimino, i.e. aminohydroxy, dimers are converted to theircorresponding phosphoramidate derivatives in the same manner as thisexample and incorporated into oligonucleotide in the standard manner asnoted below. An oligomer bearing the oxime linked nucleoside dimer isreduced to an oligomer bearing a corresponding hydroxylamine linkednucleoside dimer. As noted in other examples, reduction can be effectedas an CPG bound oligomer or after removal from the CPG.

Example 17 Synthesis of Intermittent (3′-CH═N—O—CH₂-5′), i.e. oxime;(3′-CH₂—NH—O—CH₂-5′), i.e. aminohydroxy; (3′-CH₂—N(CH₃)—O—CN₂-5′), i.e.N-methyl-aminohydroxy; (3′-CH₂—O—N(CN₃)—CH₂-5′), i.e.N-methyl-hydroxyamino; or (3′-CH₂—N(CN₃)—N(CH₃)—CH₂-5′), i.e.N,N′-dimethyl-hydrazino Linked Oligonucleosides

An appropriate 2′-deoxynucleoside that will become the 3′-terminalnucleoside of an oligonucleoside is bound to a CPG column for use on anABI 380B automated DNA synthesizer. Standard phosphoramidite chemistryprogram steps were employed to place the dimer bearing the(3′-CH═N—O—CH₂-5′), i.e. oxime; (3′-CH₂—NH—O—CH₂-5′), i.e. aminohydroxy;(3′-CH₂—N(CH₃)—O—CH₂-5′), i.e. N-methyl-aminohydroxy;(3′-CH₂—O—N(CH₃)—CH₂-5′), i.e. N-methyl-hydroxyamino; or(3′-CH₂—N(CH₃)—N(CH₃)—CH₂-5′), i.e. N,N′-dimethylhydrazino, linkagesinto the desired position or positions of choice within the sequence.

Example 18 Synthesis of Uniform (3′-CH═N—O—CH₂-5′) or(3′-CH₂—NH—O—CH₂-5′) Linked Oligonucleosides Via an ABI 380B DNASynthesizer, Utilizing 3 Nucleoside Subunits

Subunit 1: CPG-bound 5′-O-phthalimidothymidine prepared according to theprocedure of Nucleic Acids Research, 18:3813 (1990), and used as a3′-terminal unit for oligonucleoside synthesis.

Subunit 2: Bifunctional (3′-C-formyl and 5′-O-phthalimidodeoxyribonucleoside) derived by standard glycosylation of methyl3-deoxy-3-C-cyano-5-O-(phthalimido)-β-D-erythro-pentofuranoside with anappropriate base and DIBAL-H reduction of the nucleoside product.

Subunit 3: 5′-O-DMT-3′-C-formyl thymidine, employed for theincorporation of the last (the 5′-end of the oligonucleoside)nucleoside.

The automated steps of a cycle that is required to synthesize a uniformlinkage (on a 10 μM scale : loading of unit 1 on CPG) are as follows:

STEP REAGENT/SOLVENT Time/min 1 5% Methylhydrazine in DCM 10 2 DCM:MeOH(9:1, v/v) 5 3 DCM wash 2 4 3′-C-formyl-5′-O-phthalimido-deoxyribo- 3nucleoside (Unit 2, 20 μM in 20 ml of DCM) 5 DCM:Acetone (9:1, v/v):Capping 2 6 DCM wash 3Foregoing steps 1 through 6 are repeated for each addition of anucleoside unit depending on desired sequence and length. The final unitis then added:

8 Final nucleoside (20 μM in 20 ml 5 DCM) or Unit 3

Example 19 General and Specific NaBH₃CN Reduction for Conversion of(3′-CH═N—O—CH₂-5′) Linkage to (3′-CH₂—NH—O—CH₂-5′)

Reduction of a Dimer

To a solution of a dimer (0.49 g, 1 mmol) in glacial acetic acid (AcOH)(5 ml) was added sodium cyanoborohydride (0.19, 3 mmol) in AcOH (1 ml),under an argon atmosphere at room temperature. The suspension wasstirred for 1 h, and an additional amount of NaBH₃CN in AcOH (1 ml) wasadded and stirring continued for 1 h. The excess of AcOH was removedunder reduced pressure at room temperature. The residue was coevaporatedwith toluene (2×50 ml) and purified by silica gel (25 g) columnchromatography. Elution with CH₂Cl₂:MeOH (9:1, v/v) and pooling ofappropriate fractions, followed by evaporation furnished 0.36 g (75%) ofsolid dimer.

Reduction of an Oligonucleoside

CPG-bound oligonucleoside (1 μM), that contains one (or more) backbonemodified linkages is taken off the DNA synthesizer after completion ofits synthesis cycles. A 1.0 M NaBH₃CN solution in THF:AcOH (10 ml, 1:1v/v) is pumped through the CPG-bound material in a standard wayutilizing a syringe technique for 30 min. The column is washed with THF(50 ml), and the reduced oligonucleoside is released from the supportcolumn in a standard way.

Alternative Reduction of an Oligonucleoside

As an alternative to the above reduction, reduction can also beaccomplished after removal from the CPG support. At the completion ofsynthesis the oligonucleoside is removed from the CPG-support bystandard procedures. The 5′-O-trityl-on oligonucleoside is purified byHPLC and then reduced by the NaBH₃CN/AcOH/THF method as described above.

Example 20 Synthesis of (3′-CH₂—N(CH₃)—O—CN₂-5′) Linked Oligonucleosidehaving a 2′,3′-didehydro Nucleoside as its 5′ Terminal Nucleoside

3′-De(oxyphosphinico)-2′,3′-didehydro-3′-[methylene-(methylimino)]thymidylyl-(3′→5′)thymidine.

To a stirred solution of1-(5′-O-(MMTr)-β-D-glyceropentofuran-3′-ulosyl]thymine (0.13 mmol;prepared according to the procedure of T.-C. Wu, et al., Tetrahedron,45:855 (1989),5′-O-(methyleneamino)-3′-O-(t-butyldiphenylsilyl)thymidine (0.13 mmol;prepared according to the procedure of F. Debart et al. Tet. Letters,33, in press, (1992), ethylene glycol (0.5 mmol), and HMPA (0.5 ml) wasadded SmI₂ in THF (0.1 mol, 3 ml, 3 mmol) at room temperature. The colorof SmI₂ fades out as the reaction proceeds to form the desired adduct.After complete disappearance of starting materials the reaction mixtureis worked-up in the usual way. (The product could be purified by silicacolumn chromatography for characterization). The crude mixture of3′-epimeric adduct is then alkylated (HCHO/NaCNBH₃/AcOH) as described inother of these examples. The methylated product is then treated withmethylsulfonylchloride in pyridine to obtain a 3′-epimeric mesylate,which on base treatment would furnish the title compound.

Example 21 Synthesis of (3′-CH₂—CH₂—NH—CH₂-5′) Linked Oligonucleoside

3′-De(oxyphosphinico)-3′-(1,2-ethanediylimino)-thymidylyl-5′-O-(t-butyldimethylsilyl)-(3′→5′)-3′-O-(t-butyldiphenylsilyl)-5′-deoxythymidine

To a stirred solution of aldehyde [2.5 g, 6.5 mmol, freshly preparedaccording to the procedure described by Fiandor, J. and Tam, S. Y.,Tetrahedron Letts., 33:597 (1990)],5′-amino-3′-O-(t-butyldiphenylsilyl)-5′-deoxythymidine [3.13 g, 6.5mmol, prepared in two steps via 3′-O-silylation of5′-azido-5′-deoxythymidine in the manner of Hata et al. J. Chem. Soc.Perkin I, p. 306 (1980) and subsequently reduction of the product by themethod of Poopeiko et al., Syn. Lett., p. 342 (1991)], AcOH (0.39, and6.5 mmol) in dicholoroethane (65 ml) was added followed by NaBH(OAc)₃(2.759, 13.08 mmol) under argon. The suspension was stirred for 3 hoursat room temperature. The reaction mixture was diluted with CH₂Cl₂ (250ml) and washed with water (2×100 ml). The organic layer was dried(MgSO₄) and concentrated to furnish the crude product as a syrup. Theproduct was purified by silica gel column chromatography to furnish thetitle compound as white foam (3.5 g, 64%). ¹H NMR (CDCl₃) δ 0.1 [s, 6,Si(CH ₃)₂]; 0.9 and 1.1 [2s, 18, 2 Si(CH ₃)₃]; 1.85 and 1.95 (2s, 6, 2CH ₃); 2.5 (m, 2, 3″CH ₂); 3.7 (m, 2, 5′CH ₂); 4.0 (m, 2, 3′,4′CH); 4.2(m, 1, 3′CH); 6.05 (m, 1, 1′H); 6.28 (t, 1, 1′H); 7.1 and 7.57 (2s, 2,C6H); 7.35-7.7 [2m, 12, Si ArH)₂], and other sugar protons.

3′-De(oxyphosphinico)-3′-(1,2-ethanediylimino)thymidylyl-(3′→5′)-5′-deoxythymidine

The protected dimer was deblocked in 81% yield following the standardprocedure using (Bu)₄NF in THF. The deblocked dimer was purified by HPLCfor analysis. ¹H NMR (DMSO-d₆) δ1.76 and 1.78 (2s, 6, CH ₃); 2.0-2.2(3m, 4, 2′CH ₂); 3.15 (m, 2, NCH ₂); 3.56 (m, 2, 4′H, 5′CH ₂); 4.18 (brs, 1, 3′H); 5.17 and 5.22 (2 br s, 2, 2 OH); 5.95 (t, 1, 1′H); 6.1 (t,1, 1′H); 7.6 and 7.85 (2s, 2, 2(C₆ H)); 11.25 (br s, 2 2NH) and otherprotons.

Example 22 Synthesis of Bi-functional Nucleoside Alternate Method tothat of Example 18 Subunit 2

3-Deoxy-3′-C-[(methoxyimino)methyl]-thymidine

To a stirred solution of 3′-deoxy-3′-C-formyl-5′-O-tritylthymidine(0.59, 1 mmol, prepared as per Example 4 in CH₂Cl₂:MeOH (2:1, 30 vol.)was added AcOH (0.5 ml) and methoxyamine hydrochloride (0.189, 2.2 mml)at room temperature. The mixture was stirred for 30 min., concentratedunder vacuum and the residue dissolved in MeOH (20 ml). To thissolution, concentrated HCl (0.1 ml) was added and stirred for 1 h. Thesolution was neutralized with NH₄OH (≈2 ml) and concentrated under avacuum to furnish the 3′-C-[(methoxyimido)methyl] derivative ofthymidine. ¹H NMR (CDCl₃) δ 9.67 (s, 1, NH); 7.67 (s, 1, H-6); 7.33 (d,0.70, H-3″ E isomer), 6.65 (d, 0.30, H-3′ Z isomer); 6.15 (m, 1, H-1′);3.60-4.12 (m, 3.3, H-4′, H-5′5″, H-3′ Z isomer); 3.91 (s, 0.9, OCH ₃ Zisomer); 3.82 (s, 2.1, OCH ₃ oxime E isomer); 3.26 (m, 0.7, H-3′ Eisomer); 2.27-2.60 (m, 2, H-2′,2″); 1.91 (2s, 3, C₆CH ₃).

3′-Deoxy-3′-C-[(methoxyimino)methyl]-5-methylcytidine

The 5-methyl cytidine analogue was prepared in a like manner to thethymidine above. ¹H NMR (CDCl₃) δ 7.82 (s, 0.34, H-6 Z isomer), 7.75 (s,0.66, H-6 E isomer); 7.32 (d, 0.66, H-3″ E isomer, J_(3′,3″)=6.63 Hz);6.64 (d, 0.34, H-3″ Z isomer, J_(3′,−3″)=6.8 Hz); 6.12 (m, 1, H-1);3.50-4.15 (m, 3.34, H-4′, H-5′5″, H-3′ Z isomer); 3.91 (s, 1.02, OCH ₃)oxime Z isomer); 3.83 (s, 1.98, OCH ₃ oxime E isomer); 3.20 (m, 0.66,H-3′ E isomer); 2.3-2.6 (m, H-2′,2″); 1.92 and 1.94 (2s, 3, C₅CH ₃ E andZ isomers).

3′-Deoxy-3′-C-[(methoxyimino)methyl]-5′-O-phthalimidothymidine

3,-Deoxy-3′-C-[(methoxyimino)methyl]-thymidine on treatment with Ph₃P,N-hydroxyphthalimide and DEAD (Mitsunobu conditions) furnished the5′-O-phthalimidothymidine derivative. ¹H NMR (CDCl₃) δ 8.45 (br s, 1,NH); 7.4-8 (m, ≈5.64, aromatic H, H-6, C_(3″) H═N E isomer); 6.72 (d,0.36, H-3″ Z isomer); 6.15 (m, 1, H-1′); 4.4-4.65 (m, 3, H-4′, H-5′,5″);4.25 (m, 0.36, H-3′ Z isomer); 3.92 (s, 1.08, OCH ₃ oxime Z isomer);3.85 (s, 1.92, OCH ₃ oxime E isomer); 3.46 (m, 0.64, H-3′ E isomer);2.30-2.60 (m, 2, H-2′, 2″); 1.97 (2s, 3, C₅CH ₃).

3,-Deoxy-3′-C-(formylmethyloxime)-5′-phthalimido-5-methylcytidine

The 5-methyl cytidine analogue was prepared in a like manner to thethymidine above. ¹H NMR (CDCl₃) δ 7.7-7.95 (m, 5, aromatic H, H-6); 7.40(d, 0.65, H-3″ E isomer; J_(3′3″)=5.87 Hz); 6.69 (d, 0.35, H-3″ Zisomer, J_(3′,3″)=6.3 Hz); 6.16 (m, 1, H-1′); 4.35-4.70 (m, 3, H-4′,H5′,5″); 4.30 (m, 0.35, H-3′ Z isomer); 3.88 (s, 1.05, OCH ₃ Z isomer);3.81 (s, 1.95, OCH ₃ E isomer); 3.26 (m, 0.65, H-3′ E isomer); 2.30-2.65(m, 2, H-2′,2″); 2 and 1.98 (2s, C₅ H ₃Z and E isomers).

3′-Deoxy-3′-C-formyl-5′-O-phthalimidothymidine

3′-Deoxy-3′-C-[(methoxyimino)methyl]-5′-O-phthalimidothymidine upontreated with CH₃CHO in MeOH regenerated the 3′-C-formyl group. Theproduct on purification by silica gel column chromatography furnishedthe title compound as homogeneous material in 81% overall yield for 3steps. ¹H NMR (CDCl₃) δ 9.95 (s, 1, CH═O); 8.62 (br s, 1, NH); 7.71-7.90(m, 5, aromatic H, H-6); 6.06 (t, 1, H-1′, J_(1′2′)=6.61 Hz,J_(1′2′)=6.6 Hz); 4.36-4.73 (m, 3, H-4′, H-5′,5″); 3.78 (m, 1, H-3′);2.20-2.90 (m, 2, H-2′,2″); 1.98 (s, 3, C₅CH ₃).

Example 23 Synthesis of Uniform 3′-CH═N—O—CH₂-5′ or 3′-CH₂—NH—O—CH₂-5′-or 3′-CH₂—N(CH₃)—O—CH₂-5′ Linked Tetramer via Solution Phase Chemistry

3′→5′ Elongation

A standard coupling (as described in Example 12) of3′-deoxy-3′-C-formyl-5′-O-phthalimidothymidine with5′-O-amino-3′-O-(t-butyldiphenylsilyl)thymidine furnished3′-de(oxyphosphinico)-3′-(methylidynenitrilo)-thymidylyl-5′-O-phthalimido-(3′→5′)-3′-O-(t-butyldiphenylsilyl)thymidine.The latter product on the treatment with methylhydrazine (as describedin Example 11) gave 5′-O—NH₂-T-3′-CH═N—O—CH₂-5′-T-3′-O-TBDPSi, which onanother round of coupling with 5′-O-Phth-T-3′-CHO gave the trimmer5′-O-Phth-T-3′—CH═N—O—CH₂-5′-T-3′-CH═N—O—CH₂-5′-T-3′-O-TBDPSi in anoverall 83% yield. The tetramer was reduced according to Example 14using NaBH₃CN/AcOH to furnish5′-O-Tr-T-3′-CH₂NH—O—CH₂-5′-T-3′-CH₂—NH—O—CH₂-5′-T-3′-CH₂—NH—O—CH₂-5′-T-O-3′-TBDPSi.The reduced tetramer on further reductive alkylation usingHCHO/NaBH₃CN/AcOH gave5′-O-Tr-T-3′-CH₂—N(CH₃)—O—CH₂-5′-T-3′-CH₂—N(CH₃)—O—CH₂-5′-T-3′-CH₂—N(CH₃)—O—CH₂-5′-T-3′-O-TBDPSi.The methylated tetramer was finally deblocked using HCl and n(Bu)₄NFtreatments to yield free tetramer5′-OH-T-3′-CH₂—N(CH₃)—O—CH₂-5′-T-3′-CH₂—N(CH₃)—O—CH₂-5′-T-3′-CH₂—N (CH₃)—O—CH₂-5′-T-3′-OH in 79% overall yield. The tetramer was purified byHPLC utilizing a reverse phase column (Supelcosil LC18 5_(μ), 15 cm×4.5cm) and elution with H₂O→CH₃CN (H₂O:CH₃CN, 95:5, 1 min; 8:2, 20 min;7:3, 30 min; 2:8; 40 min/ml) gradient. The tetramer was eluted after26.96 min as single isolated peak that corresponded to 86% of the totalproducts eluted. The fractions at 26-27.5 min were pooled andlyophilized to furnish white powder. The exact molecular weight of thetetramer was determined by MS FAB: m/z 1045 (M+H)⁺. As noted above, forthe NMR data the rings are numbered from the 5′ terminus to the 3′terminus. ¹H NMR (D₂O, 40° C.) TOSEY (30, 100 M sec Mix)

Unit T₄ H-1′ 6.36 H-2′, 2″ 2.53 H-3′ 4.55 H-4′ 4.22 H-5′, 5″ 4.04, 4.18H-6 7.05 Unit T₃ H-1′ 6.22 H-2′ 2.52 H-2″ 2.71 H-3′ 2.90 H-3″ 2.91, 2.97H-4′ 4.12 H-5′, 5″ 4.04, 4.23 H-6 7.18 C₅CH₃ 1.88 Unit T₂ H-1′ 6.23 H-2′2.52 H-2″ 2.71 H-3′ 2.91 H-3″ 2.91, 2.97 H-4′ 4.12 H-5′, 5″ 4.04, 4.23H-6 7.14 C₅CH₃ 1.88 Unit T₁ H-1′ 6.26 H-2′ 2.54 H-2″ 2.73 H-3′ 3.03 H-3″3.03, 2.93 H-4′ 4.06 H-5′, 5″ 4.05, 3.90 H-6 7.26 C₅CH₃ 1.90 N-CH₃backbone broad 2.83

The above solution phase reactions can be easily transferred to an ABI380B DNA synthesizer, utilizing 3-nucleoside sub units as describedabove.

Example 24 Synthesis of Monomer Unit for (3′-CH₂—O—N═CH-5′),(3′-CH—O—NH—CH₂-5′) and (3′-CH₂—O—N(CH₃)—CH₂-5′) Linkages

1-[3′-Deoxy-3′-C-(hydroxymethyl)-5′-O-(trityl)-β-D-erythropentofuranosyl]-thymine

A suspension of NaBH₄ (1.36 g, 9.6 mmol) was added dropwise to a stirredsolution of 3′-C-formyl-5′-O-tritylthymidine in EtOH:H₂O (22 ml, 3:1,v/v) mixture at room temperature. After 3 h, EtOAc (300 ml) was addedand the organic layer was washed with H₂O (2×150 ml). The dried (MgSO₄)EtOAc extract was evaporated under reduced pressure and the residue waspurified by silica gel column chromatography. Elution with CH₂Cl₂:MeOH(9:1, v/v), pooling and concentration of appropriate fractions gave thetitle compound (1.13 g, 83%). ¹H-NMR (CDCl₃) δ 8.29 (br s, 1, NH), 7.59(s, 1, C₆H) 7.47-7.22 (m, 15, TrH) 6.13 (dd, 1, H _(1′), J_(1′,2′)=6.5Hz); 3.98 (m, 1, H _(4′)); 3.62 (m, 2, H _(3′)), 3.56-3.33 (m, 2, H_(5′),H _(5″)), 2.60 (m, 1, H _(3′)); 2.33-2.20 (m, 2, H _(2′) H _(2′));1.91 (br s, 1, OH); 1.53 (S, 3, CH ₃).

1-[3′-Deoxy-3′-C-[O-(phthalimidohydroxymethyl)]-5′-O-trityl-β-D-erythro-pentofuranosyl]-thymine

Diisopropylazodicarboxylate (0.47 ml, 2.41 mmol) was added to a stirredsolution of 3′-deoxy-3′-C-(hydroxymethyl)-5′-O-trityl-thymidine (0.8 g,1.62 mmol), N-hydroxyphthalimide (0.35 g, 2.15 mmol), triphenylphosphine(0.56 g, 2.15 mmol) in dry THF (10 ml) at room temperature. After 48 h,the products were concentrated and the residue was extracted with CH₂Cl₂(2×100 ml). The CH₂Cl₂ extracts were washed with NaHCO₃ (5%, 100 ml) andwater (100 ml). The dried (MgSO₄) extract was evaporated under reducedpressure and the residue was purified by short-silica gelchromatography. Elution with EtOAC:Hexanes (1:1, v/v), pooling andconcentration of appropriate fractions gave the title compound as whitefoam (0.82 g, 79%). ¹H-NMR (CDCl₃) δ 8.24 (s, 1, NH); 7.85-7.20 (m, 2O,TrH, ArH, C₆ H), 6.20 (m, 1, H _(1′)), 4.22-4.16 (m, 3, H _(4′), H_(3″)), 3.63-3.40 (m, 2, H _(5′), H _(5″)), 3.02 (m, 1, H _(3′)),2.50-2.43 (m, 2, H _(2′) H _(2″)); 1.51 (s, 3, CH ₃). Anal. Calcd. forC₃₈H₃₃N₃O₇. 0.5 EtOAc:C, 69.86; H, 5.42, N, 6.11. Found: C, 70.19; H,5.27; N, 5.75

1-{3′-Deoxy-3′-C-[O-(aminohydroxymethyl)]-5′-O-trityl-β-D-erythro-pentofuranosyl}-thymine

Methylhydrazine (0.12 ml, 2.25 mmol) was added to a stirred solution of3′-deoxy-3′-C-[O-(phthalimidohydroxymethyl)]-5′-O-tritylthymidine (0.77g, 1.2 mmol) in day CH₂Cl₂ (9 ml) at room temperature. After 1 h, theprecipitate was filtered and the residue washed with CH₂Cl₂ (2×10 ml).The combined filtrates were concentrated and the residue was purified bysilica gel column chromatography. Elution with CH₂Cl₂:MeOH (97:3, v/v),pooling and evaporation of appropriate fractions gave the title compoundas white powder (0.43 g, 70%). ¹H-NMR (CDCl₃) δ 8.59 (br s, 1, NH), 7.66(m, 1, C₆H), 7.40-7.15 (m, 15, TrH), 6.06 (pseudo t, 1, H _(1′)), 5.22(br s, 2, NH ₂), 3.89 (m, 1, H _(4′)), 3.65-3.20 (m, 4, H _(5′), H_(5″), H _(3″)), 2.81 (m, 1, H _(3′)), 2.21-2.13 (m, 2, H _(2′), H_(2″)), 1.37 (s, 3, CH ₃).

Example 25 Synthesis of (3′-CH₂—O—N═CH-5′), (3′-CH₂—O—NH—CH₂-5′) and(3′-CH₂—O—N(CH₃)—CH₂-5′) Linked Oligonucleosides

3′-De(oxyphosphinico)-3′-[methyleneoxy(methylimino)]thymidylyl-(3′→5′)-5′-deoxythymidine

A mixture of1-[4-C-formyl-3-O-(t-butyldiphenylsilyl)-β-D-erythro-pentofuranosyl)thymine(1 mmol, prepared according to the procedure of Nucleosides andNucleotides, 9:533 (1990)],3′-deoxy-3′-C-[(O-(aminohydroxymethyl)]-5′-O-tritylthymidine (1 mmol),AcOH (0.1 ml), and dry CH₂Cl₂ (25 ml) was stirred at room temperaturefor 1 h. The solvent was evaporated and the residue was dissolved inglacial AcOH (5 ml). NaBH₃CN (3 mmol) was added to the stirred AcOHreaction mixture. After 1 h, an additional amount of NaBH₃CN (3 mmol)was added and the mixture stirred for 1 h. The reaction was concentratedunder vacuum and the residue was purified by silica gel columnchromatography to furnish 5′-O-Tr-T-3′-CH₂—O—NH—CH₂-5′-T-3′-O-TBDPSidimer. ¹H-NMR (CDCl₃) δ 8.73 (br s, 2, 2NH), 7.67 (s, 1 C₆H), 7.674-7.23(m, 20, TrH, TBDPhH), 6.96 (s, 1, C₆ H), 6.23 (pseudo t, 1, T₂ H _(1′)),6.11 (pseudo t, 1, T₁, H _(1′)) 5.51 (br s, 1, NH), 4.16 (m, 1, T₂ H_(3′)) 4.02 (m, 1, T₂ H _(4′)), 3.87 (m, 1, T₁ H _(4′)), 3.52 (m, 3, T1CH _(23″), T₁ H _(5″)), 3.23 (m, 1, T₁ H _(5′)), 2.55-2.76 (m, 3, T1 H_(3′), T2 H _(5′) H _(5″)) 2.33-2.27 (m, 1, T2 H _(2′)), 2.23-2.12 (m,2, T1 H2 H _(2″)), 1.95-1.85 (m, 1, T₂ H _(2″)), 1.83 (s, 3, CH ₃) 1.45(s, 3, CH ₃), 1.06 (s, 9, (CH ₃)₃CSi).

The latter dimer was methylated using HCHO/NaBH₃CN/AcOH and finallydeblocked with nBu₄NF/THF and HF/CH₃CN in two-steps to furnish the titlecompound (65% yield). ¹H-NMR (DMSO-d₆) δ 11.27 (br s, 2, NH); 7.85 (s,1, T1 C₆H); 7.51 (s, 1, T₂ C₆ H); 6.15 (pseudo t, 1, T₂ H ₁,J_(1′-2′)=7.8 Hz, J_(1′-2″)=6.3 Hz); 6.00 (pseudo t, 1, T₁ H _(1′),J_(1′-2′)=6.9 Hz, J_(1′-2″)=4.5 Hz), 5.32 (br s, 1, OH _(3′)), 5.09 (brs, 1, OH _(5′)) ; 4.17 (m, 1, T₂ H _(3′)); 3.90 (m, 1, T₂ H ₄);3.76-3.66 (m, 4, T₁ H _(4′), T₁ H _(5′), CH _(2 3′)) ; 3.60-3.52 (m, 1,T₁ H _(5″)); 2.82 (m, 2, T₂ H _(5′), H _(5″)) ; 2.57 (s, 3, N—CH ₃);2.47 (m, 1, T₁ H _(3′)); 2.23-2.02 (m, 4, H _(2′) H _(2″)) 1.81 (s, 3,C₅CH ₃); 1.78 (s, 3, C₅ CH ₃). Anal. Calcd. for C₂₂H₃₁N₅O₉.O.5 H₂O: C,50.96; H. 6.22; N, 13.50. Found: C, 51.01; H, 6.22; N, 13.19. MS (FAB+,glycerol) M+H⁺ m/z=510.

Example 26 Synthesis of Phosphoramidate Containing(3′-CH₂—O—N(CH₃)—CH₂-5′) Linked Oligonucleoside

3′-De(oxyphosphinico)-3′-[methyleneoxy(methylimino)]-thymidylyl-5′-O-(dimethyoxytriphenylmethyl)-(3′→5′)-3′-(O-β-cyanoethyldiisopropylaminophosphiryl)thymidine

Dimethyoxytritylation of the dimer 5′-OH-T-3′-CH₂—O—NCH₃—CH₂-5′-T-3′-OHfollowing the procedure described in oligonucleotide Synthesis: apractical approach, Ed. M. J. Gait, IRL Press, 1984, furnished the5′-O-DMTr protected dimer as white foam. ¹H-NMR (CDCl₃) δ 7.67 (s, 1, H₆); 7.44-6.82 (m, 14, H ₆, DMTrH); 6.20 (pseudo t, 2, H _(1′)); 4.3 (m,1, T₂ H _(3′)); 4.15 (m, 1, T₂ H _(4′)); 4.00 (m, 1, T₁ H _(4′)); 3.80(s, 6, OCH ₃); 3.77-3.23 (m, 4, T₁ H _(5′) H _(5″), CH_(2 3″));2.89-2.50 (m, 3, T₂ H _(5′) H _(5″), T1 H ₃′); 2.62 (s, 3, N—CH ₃);2.48-2.08 (m, 4, H _(2′) H _(2″)); 1.9 (s, 3, C₅CH ₃) 1.48 (s, 3 C₅CH₃).

Above compound was phosphitylated following the procedure described inOligonucleotide Synthesis: a practical approach, Ed. M. J. Gait, IRLPress, 1984, to furnish the title compound in 70% yield over two stepsas white powder. ¹H NMR (CDCl₃) δ 8.25 (br s, 2, NH), 7.66 (s, 1, C₆ H),7.15-7.45 (m, 10, ArH, C₆ H), 6.8-6.9 (m, 4, ArH), 6.12 (m, 2, 2C_(1′)H), 3.79 (s, 6, ArOCH ₃), 2.56 (s, 3, N—CH ₃), 1.88, 1.44 (2s, 6, 2 C₅CH ₃) and other protons. ³¹P NMR (CDCl₃) 149.42 and 148.75 ppm.

Example 27 Synthesis of Oligonucleosides having Linkages that IncludePharmacokinetic and Pharmacodynamic Property Modifying Groups LocatedTherein On

3′-De(oxyphosphinico)-3′-[methylene(benzylimino)]-thymidylyl-5′-O-(dimethyoxytriphenylmethyl)-(3′→5′)-3′-O-β-(cyanoethyldiisopropylaminophosphiryl)thymidine

A reductive coupling of 3′-deoxy-3′-C-formyl-5′-O-tritylthymidine (1.5mmol) with 5′-O-amino-3′-O-(t-butyldiphenylsilyl)thymidine (1.5 mmol) asdescribed in Example 12 furnished5′-O-Tr-T-3′-CH₂—NH—O—CH₂-5′-T-3′-O-TBDPSi dimer. This dimer wasbenzylated with C₆H₅CHO/NaBH₃CN/AcOH in the same manner as the abovedescribed methylation to yield N-benzylated dimer5′-O-Tr-T-3′-CH₂-NBz-O—CH₂-5′-T-3′-O-TBDPSi. The latter dimer wasdeblocked using nBu₄NF/THF and HCl/MeOH methodology as described inabove examples to furnish deblocked dimer5′-OH-T-3′-CH₂-NBn-O—CH₂-5′-T-3′-OH, which on dimethoxytritylation andsubsequent phosphitylation following the procedure described inOligonucleotide Synthesis: a practical approach, Ed. M. J. Gait, IRLPress, 1984, gave the title compound (45% overall yield). ¹H NMR (CDCl₃)δ 6.15 (pseudo t, 1, T2 C_(1′) H); 6.09 (m, 1, T1 C_(1′) H); 3.76 (s, 6,2OCH ₃); 1.7 and 1.48 (2S, 6, 2-CH ₃) and other protons. ³¹p NMR (CDCl₃)149.59 and 149.23 ppm.

The phosphiltylated dimer was successfully incorporated into an oligomerusing an automated DNA synthesizer in the manner of Example 8illustrating the ability to attach of various pharmacokinetic andpharmacodynamic property modifying groups into the backbone linkageprior to the DNA synthesis of an oligonucleotide.

Example 28 Of (3′-CH₂—NH—CH₂—CH₂-5′), (3-CH₂—N(CH₃) —CH₂—CH₂-5′), andPhosphoramidate Derivative

3′-De(oxyphosphinico-3′-[(methyleneimino)methylene]-5′-O-(dimethyoxytrityl)thymidylyl-(3′→5′)-thymidine

A reductive amination [according to the procedure of A. F. Abdel-Magidet al., Tetrahedron Letts. 31:5595 (1990)] of3′-deoxy-3′-C-formyl-5′-O-tritylthymidine (1 mmol) with1-[6′-amino-2′,5′,6′-trideoxy-3′-O-(t-butyldiphenylsilyl)-β-D-erythro-hexofuranosyl]thymine[1.2 mmol, prepared according to the procedure of G. Et Zold et al.,J.C.S. Chem. Comms., 422 (1968)] in presence of AcOH gave a blockeddimer 5′-O-Tr-T-3′-CH₂NH—CH₂—CH₂-5′-T-3′-O-TBDPSi, which on deprotectionas described in above examples gave 5′-OH-T-3′-CH₂—NH—CH₂—CH₂-5′-T-3′-OHdimer as white powder (70% yield). ¹H NMR (D₂O), pH 5.6, 20° C.) δ T1thymidine unit: 7.78 (s, 1, C₆ H); 6.17 (t, 1, C₁ H); 4.45 (m, 1, C_(3′)H); 4.08 (m, 1, C_(4′) H); 4.00, 3.72 (m, 2, C_(5′,5″) H); 2.9 (m, 2C_(6′,6″) H); 2.34 (m, 2, C_(2′,2) H); 1.77 (s, 3, CH ₃); T2 thymidineunit: 7.47 (s, 1 C₆ H); 6.07 (t, 1, C_(1′) H); 3.89 (m, 2, C_(5′5″) H);3.79 (m, 1, C_(4′) H); 2.89 (m, 1, C_(3″) H); 2.38 (m, 1, C_(2′) H);2.32 (m, 1, C_(3′) H); 1.72 (s, 3, CH ₃); and 2.68 (s, N—CHhd 3).

Pka Determination:

The sensitivity of the proton chemical shift of the N-Me group of theforegoing dimer to change in response to change in pH was measured byNMR as an indicator of the pka of the backbone amine. The chemical shiftmoved downfield as the amino group was protonated. A 4 mg sample of5′-OH-T-3′-CH₂—NCH₃—CH₂—CH₂-5′-T-3′-OH dimer was dissolved in 0.6 ml of30 mM bicarbonate buffer. The pH was varied between 5.1 and 10.0 using0.1 N NaOH in 6-steps. The chemical shift of the N-methyl proton variedbetween 2.26 and 2.93 ppm, giving rise to a pka of 7.8±0.1. While we donot wish to be bound by theory, it is thus believed that atphysiological pH this backbone will be protonated.

3′-De(oxyphosphinico-3′-[methylene(methylimino)methylene]-5′-O-(dimethyoxytrityl)-thymidylyl-(3′→5′)-3′-O-(β-cyanoethyldiisopropylaminophosphiryl)thymidine

The proceeding diner was methylated using HCHO/NaBH₃CN in AcOH tofurnish 5′-OH-T-3′-CH₂—N(CH₃)—CH₂—CH₂-5′-T-3′-OH dimer, which ondimethoxytritylation and phosphitylation following the proceduredescribed in Oligonucleotide Synthesis: a practical approach, Ed. M. J.Gait, IRL Press, 1984, gave the title compound as foam (68% yield). ¹HNMR (CDCl₃) δ 6.12 (m, 2, 2C_(1′) H) ; 2.15, 2.14 (2s, 3, N—CH ₃); 1.88,1.45 (2s, 6, 2 C₅CH ₃) and other protons. ³¹P NMR (CDCl₃) 149.49 and148.96 ppm.

Example 29 A (3′-CN₂—N(Labile Blocking Group)-O—CH₂-5′) Dimer andPhosphoramidate Derivative—a Dimer Incorporating a3′-de(oxyphosphinico)-3′-(methyleneimino) (3→5′) Linkage Having a LabileN-protecting Group for Regeneration of a (3′-CH₂—NH—O—CH₂-5) Linkage

3′-De(oxyphosphinico)-3′-[methylene(phenoxyacetylimino)]-thymidylyl-(3′→5′)-thymidine

To a stirred solution of 5′-O-Tr-T-3′-CH₂—NH—O—CH₂-5′-T-3′-O-TBDPSi (1mmol, prepared according to the procedure of F. Debart et al.Tetrahedron Letts., 33:in press 1992) in dry pyridine (10 ml) was addedphenoxyacetylchloride (1.2 mmol). After 12 h, the products were dilutedwith CH₂Cl₂ (200 ml) and washed with sat. NaHCO₃ (2×50 ml), water (2×50ml) and dried (MgSO₄). The CH₂Cl₂ extract was concentrated and residuepurified by silica gel column chromatography. Elution with CH₂Cl₂: MeOH(9:1, v/v), pooling of appropriate fractions and evaporation furnished5′-O-Tr-T-3′-CH₂—N(COCH₂OPh)—O—CH₂-5′-T-3′-O-TBDPSi dimer as white foam.¹H NMR (DMSO-d ₆) δ 11.35 (br s, 2, NH); 7.6-6.65 (m, 32, Tr, TBDPS,phenoxyacetyl, C₆ H); 6.3 (pseudo t, 1, H _(1′)); 6.03 (pseudo t, 1, H_(1′)); 4.5 (m, 2, CH ₂); 4.3 (m, 1, T₂ H ₃); 3.9-3.3 (m, 6, T₁ H _(4′),T₂ H _(4′), T₂ H _(4′), T₂H_(5′) H_(5″), CH_(2 3″)); 3.10 (m, 2, T, H_(5′) H _(5″)); 2.65 (m, 1, T₁ H _(3′)); 2.2-2.05 (m, 4, H _(2′) H_(2″)); 1.58 (s, 3, CH ₃); 1.4 (s, 3, CH ₃); 1.02 (s, 9, (CH ₃)₃CSi).

The foregoing dimer was sequentially deblocked with HF (48%)/CH₃CN(5:95, v/v) treatment to remove the trityl group, and the product ontreatment with nBu₄NF/THF removed the silyl group to furnish titlecompound as white powder (70% yield for 3-steps). ¹H NMR (DMSO-d ₆) δ11.35 (br s, 1, NH); 11.25 (br s, 1, NH) 7.92 (s, 1, C₆ H); 7.5 (s, 1,C₆ H); 7.2-6.8 (m, 5, ArH); 6.23 (pseudo t, 1, H _(1′)); 5.98 (dd, 1, H_(1′)); 5.45 (d, 1, OH _(3′)), 5.15 (t, 1, OH _(5′)); 4.9 (m, 2, CH ₂);4.3-3.5 (m, 9, T₂ H _(3′), H _(4′), H _(5′) H _(5″), CH _(23″)); 2.6 (m,1, T₁ H _(3′)); 2.25-2.00 (m, 4, H _(2′) H _(2″)); 1.75 (s, 3, CH ₃);1.65 (s, 3, CH ₂).

The latter dimer was dimethoxytritylated as per the procedure ofdescribed in Oligonucleotide Synthesis: a practical approach, Ed. M. J.Gait, IRL Press, 1984, to furnish5′-O-DMT-T-3′-CH₂—N—(COCH₂OPh)—O—CH₂-5′-T-3′-OH as pale yellow coloredfoam. ¹H NMR (DMSO d₆) δ 11.3 (br s, 2, NH); 7.55 (s, 1, C₆ H). 7.45 (s,1, C₆ H); 7.38-6.75 (m, 18, DMTrH, phenoxyacetyl-H); 6.22 (pseudo t, 1,T₂ H _(1′)); 6.05 (pseudo t, 1, T₁ H _(1′)); 4.75-4.60 (m, 2, CH ₂);4.25 (m, 1, T₂ H _(5′)); 4.18 (m, 1, T₂ H _(3′)); 4.05 (m, 1, T₂ H_(5″)); 3.9 (m, 2, H _(4′)); 3.8-3.6 (m, 2, CH _(2 3″)); 3.65 (s, 6,2OCH ₃) 3.2 (m, 2, T₁, H _(5′) H _(5″)) 2.82 (m, 1, T₁ H _(3′));2.3-2.05 (m, 4, H _(2′) H _(2″)); 1.6 (s, 3, T₂ CHCH₃); 1.38 (s, 3, T1CH ₃).

The above dimer on phosphitylation following the procedure described inOligonucleotide Synthesis: a practical approach, Ed. M. J. Gait, IRLPress, 1984, furnished the phosphoramidate derivatized dimer(appropriate for use on DNA synthesizer) as a foam (75% in 2 steps). ¹HNMR (CDCl₃) δ 7.62 (s, 1, C₆ H); 7.2-7.45 (2m, 12, ArH); 6.77-7.05 (3m,7, ArH, C₆ H); 6.15 (pseudo t, 1, C_(1′) H); 6.05 (t, 1, C_(1′) H); 4.7(m, 2, 2C_(4′) H); 3.74 (2s, 6, 2ArOCH ₃); 2.95 (m, 1, C_(3′) H); 1.78,1.77 (2s, 3, C₅CH ₃); 1.41 (s, 3, C₅CH ₃), and other protons. ³¹P NMR(CDCl₃) 1.49.76 and 149.56 ppm.

Example 30 Regeneration of (3′-CH₂—NH—O—CH₂-5′) Linkage from(3′-CH₂—N(Labile Blocking Group)—CH₂—CH₂-5′) Linkage in anOligonucleotide

The phosphitylated dimer of Example 29 will be incorporated within anoligonucleotide as per the procedure of Example 8. After completion ofthe oligonucleotide on the support, the oligonucleotide is cleaved fromthe support utilizing standard ammonium hydroxide conditions. Concurrentwith the cleavage from the support the ammonium hydroxide treatment willfurther cleave the phenoxyacetyl blocking group from the imino nitrogenof the incorporated (3′-CH₂—N(COCH₂OPh)—O—CH₂-5′) oligonucleoside dimerto yield the (3′-CH₂—NH—O—CH₂-5′) linked oligonucleoside dimer withinthe oligonucleotide structure.

Example 31 Synthesis of (3′-CH₂—P(O)₂—O—CH₂-5′) and(3′-CH₂—O—P(O)₂—CH₂-5′) Linked Oligonucleosides

Synthesis of 3′-C-phosphonate Dimer

3′-hydroxymethyl-5′-O-(t-butyldiphenylsilyl)thymidine will be convertedinto its bromide by treatment with NBS. The bromide is subjected to anArbuzov reaction to furnish the phosphonate diester. Cleavage of thephosphonate diester with trimethylbromosilane gives the free acid whichon treatment with 3′-O-(t-butyldiphenylsilyl)thymidine and DCC inpyridine yields the dimer.

Synthesis of 3′-C-phosphonate Linked Oligonucleosides

The above dimer will be incorporated into an oligonucleoside by suitablyprotecting and activating the dimer as the 5′-O-DMT and3′-O-phosphoramide derivative for insertion into desired locations inoligonucleosides by standard DNA synthesizer chemistry.

Synthesis of 5′-C-phosphonate Linked Oligonucleosides

The corresponding 5′-C-phosphonate dimers will be obtained by a reactinga 5′-deoxy-5′-bromonucleoside with a phosphite ester resulting in a5′-phosphonate. This in turn is reacted with a 3′-hydroxymethylnucleoside to yield the 5′-C-phosphonate linked dimer.

Evaluation

Procedure 1—Structure and Integrity of Oligonucleotides

A. Digest of Oligonucleotides

Enzymatic Digestion of Oligonucleotides

The incorporation of backbone modification as in various antisenseoligonucleotides was proved by enzymatic hydrolysis using followingprotocol. In the sequence listing of this procedure a “*” is used todenote the positioning of a linkage of the invention within the sequenceand in a like manner a “p” is used to denote the positioning of a normalphosphodiester linkage.

The modified oligonucleotide

-   -   (5′-GpCpGpTpTpTpTpT*TpTpTpTpTpGpCpG-3′) (0.2 OD at A₂₆₀ ^(nm))        was dissolved in 0.1M tris-HCl buffer (pH 8.3 200 μl) and        treated with snake venom phosphodiesterase (0.4 μg), alkaline        phosphatase (0.4 μg), and calf spleen phosphodiesterase (0.4 μg)        for 24-60 h at 37° C. The resulting mixture was diluted and        analyzed by HPLC. Column: C-18 Nucleosil (5 μ). Flow rate: 1        ml/min. Solvent A: 10 mM triethylammonium acetate, Solvent B:        acetonitrile/water (1:1). A 20 min. linear gradient from 0% B to        50% B. Quantification of the material was made on the basis of        the peak areas which were directed by the extinction        coefficients of the nucleoside constituents. The identity of        each modified backbone containing dimer was proved by        co-injecting a synthetic sample with fully digested        oligonucleotide. In all cases, integration of the peaks of HPLC        analyses demonstrated the correct gross composition of the        digested oligonucleotide.        B. Integrity of Backbone Linkage

In addition, the integrity of each incorporation of modified backbonewas further supported by ¹H and ³¹p NMR analyses of a CpT*TpG tetramerprepared on the same ABI 380B DNA synthesizer. Thus, indirectlyvalidating the computer program on the synthesizer.

Procedure 2—Hybridization Analysis.

The relative ability of an oligonucleotide, an oligonucleotide analogueor oligonucleoside of the invention to bind to complementary nucleicacids can be compared by determining the melting temperature of aparticular hybridization complex. The melting temperature (T_(m)), acharacteristic physical property of complementary nucleic acids, denotesthe temperature in degrees centigrade at which 50% double helical versuscoil (unhybridized) forms are present. T_(m) is measured by using the UVspectrum to determine the formation and breakdown (melting) ofhybridization. Base stacking, which occurs during hybridization, isaccompanied by a reduction in UV absorption (hypochromicity).Consequently a reduction in UV absorption indicates a higher T_(m). Thehigher the T_(m), the greater the strength of the binding of thestrands. Non-Watson-Crick base pairing has a strong destabilizing effecton the T_(m). Consequently, absolute fidelity of base pairing isnecessary to have optimal binding of an antisense oligonucleotide oroligonucleoside to its targeted RNA.

A. Evaluation of the Thermodynamics of Hybridization of OligonucleotideAnalogues.

The ability of selected oligonucleotide analogues of the invention tohybridize to their complementary RNA or DNA sequences was determined bythermal melting analysis. The RNA complement was synthesized from T7 RNApolymerase and a template-promoter of DNA synthesized with an AppliedBiosystems, Inc. 380B nucleic acid synthesizer. The RNA species ispurified by ion exchange using FPLC (LKB Pharmacia, Inc.). Antisenseoligonucleotide analogues are added to either the RNA or DNA complementat stoichiometric concentrations and the absorbance (260 nm)hyperchromicity upon duplex to random coil transition monitored using aGilford Response II spectrophotometer. These measurements are performedin a buffer of 10 mM Na-phosphate, pH 7.4, 0.1 mM EDTA, and NaCl toyield an ionic strength of either 0.1 M or 1.0 M. Data can be analyzedby a graphic representation of 1/T_(m) vs ln[Ct], where [Ct] is thetotal oligonucleotide concentration.

The results of thermodynamic analysis of the hybridization of selectedoligonucleotide analogues of the invention are shown in Table 1. In thesequence listing of this table a “*” is used to denote the positioningof a linkage of the invention within the sequence and in a like manner a“p” is used to denote the positioning of a normal phosphodiesterlinkage. Further in this table and in following tables various backbonelinkages of the invention are cross referenced between generic chemicalnames and short hand structures as follows: (3′-CH═N—O—CH₂-5′) isdenoted as oxime; (3′-CH₂—NH—O—CH₂-5′) is denoted as aminohydroxy;(3′-CH₂—N(CH₃)—O—CH₂-5′) is denoted as N-methyl-aminohydroxy;(3′-CH₂—O—N(CH₃)—CH₂-5′) is denoted as N-methyl-hydroxyamino; and(3′-CH₂—N(CH₃)—N(CH₃)—CH₂-5′) is denoted as N,N′-dimethylhydrazino.

TABLE 1 DUPLEX STABILITY (DNA-RNA) * BACKBONE T_(m) ° C. ΔT_(m) ° C.SEQUENCE = 5′- GpCpGpTpTpTpTpT*TpTpTpTpTpGpCpG -3′ Natural 50.2N-Methyl-Hydroxyamino 48.9 −1.3 N-Methyl-Aminohydroxy 49.4 −0.8N,N′-Dimethyl-Hydrazino 48.3 −1.9 Aminohydroxy 47.8 −2.4 SEQUENCE = 5′-GpCpGpTpTpTpT*TpT*TpTpTpTpGpCpG -3′ Natural 50.2 N-Methyl-Hydroxyamino47.5 −2.7 N-Methyl-Aminohydroxy 49.7 −0.5 N,N′-Dimethyl-Hydrazino 48.6−1.6 Aminohydroxy 43.7 −6.4 SEQUENCE = 5′-GpCpGpTpTpT*TpT*TpT*TpTpTpGpCpG -3′ Natural 50.2 N-Methyl-Hydroxyamino44.2 −6.0 N-Methyl-Aminohydroxy 48.2 −1.9 N,N′-Dimethyl-Hydrazino 49.0−1.2 Aminohydroxy 45.3 −4.9 SEQUENCE = 5′-GpCpGpT*TpTpTpT*TpTpTpT*TpGpCpG -3′ Natural 50.2 N-Methyl-Aminohydroxy47.8 −2.4 SEQUENCE = 5′- GpCpGpTpT*TpT*TpT*TpT*TpTpGpCpG -3′ Natural50.2 N-Methyl-Hydroxyamino 42.3 −7.9 Aminohydroxy 45.5 −4.7 SEQUENCE =5′- GpCpGpT*TpTpT*TpT*TpTpT*TpGpCpG -3′ Natural 50.2N-Methyl-Aminohydroxy 47.9 −2.3 N,N′-Dimethyl-Hydrazino 47.3 −2.8Aminohydroxy 43.9 −6.3 SEQUENCE = 5′- GpCpGpT*TpT*TpT*TpT*TpT*TpGpCpG-3′ Natural 50.2 N-Methyl-Hydroxyamino 40.0 −10.2 N-Methyl-Aminohydroxy50.8 +0.64 N,N′-Dimethyl-Hydrazino 51.3 +1.1 Aminohydroxy 44.2 −6.0SEQUENCE = 5′- CpTpCpGpTpApCpCpT*TpTpCpCpGpGpTpCpC -3′ Natural 63.4Oxime 60.2 −3.2 N-Methyl-Aminohydroxy 64.9 +1.5 N,N′-Dimethyl-Hydrazino64.9 +1.5 Aminohydroxy 62.9 −0.5 SEQUENCE = 5′-CpTpCpGpTpApCpT*TpT*TpCpCpGpGpTpCpC -3′ Natural 56.7N-Methyl-Hydroxyamino 54.3 −2.4 N-Methyl-Aminohydroxy 57.4 +0.7N,N′-Dimethyl-Hydrazino 57.0 +0.3 Aminohydroxy 56.0 −0.7 SEQUENCE = 5′-CpGpApCpTpApTpGpCpApApTpT*TpC -3′ Natural 44.1 Oxime 41.6 −2.5N-Methyl-Hydroxyamino 43.8 −0.3 N-Methyl-Aminohydroxy 43.6 −0.5N,N′-Dimethyl-Hydrazino 42.8 −1.3 Aminohydroxy 43.4 −0.7

In a further study, the base pair specificity of oligonucleotide havingmodified linkages of the invention was studied. The study measurebinding of the 5′-T of T*T dimer in the sequence

-   -   5′-CpTpCpGpTpApCpCpT*TpTpCpCpGpGpTpCpC-3′ when matched to A in        the RNA complement (a T:rA pair) as compared to mismatch with C,        G or U. The average of the mismatch of all the base pairs is        shown in Table 2. Table 2 demonstrates that the essential        Watson-Crick base pair specificy of the backbone linkages of the        invention to complementary strand is not compromised.

TABLE 2 BASE PAIR SPECIFICITY 5′- CpTpCpGpTpApCpCpT*TpTpCpCpGpGpTpCpC-3′ * BACKBONE ΔT_(m)/mismatch Natural −5.5 Oxime −4.95N-Methyl-Aminohydroxy −7.32 N,N′−Dimethyl-Hydrazino −7.41 Aminohydroxy−6.89B. Fidelity of Hybridization of Oligonucleotide Analogues

The ability of the antisense oligonucleotide analogues of the inventionto hybridize with absolute specificity to a targeted mRNA can be shownby Northern blot analysis of purified target mRNA in the presence oftotal cellular RNA. Target mRNA is synthesized from a vector containingthe cDNA for the target mRNA located downstream from a T7 RNA polymerasepromoter. Synthesized mRNA is electrophoresed in an agarose gel andtransferred to a suitable support membrane (i.e. nitrocellulose). Thesupport membrane is blocked and probed using [³²P]-labeledoligonucleotide analogues. The stringency is determined by replicateblots and washing in either elevated temperatures or decreased ionicstrength of the wash buffer. Autoradiography is performed to assess thepresence of heteroduplex formation and the autoradiogram quantitated bylaser densitometry (LKB Pharmacia, Inc.). The specificity of hybridformation is determined by isolation of total cellular RNA by standardtechniques and its analysis by agarose electrophoresis, membranetransfer and probing with the labelled oligonucleotide analogues.Stringency is predetermined for an unmodified antisense oligonucleotideand the conditions used such that only the specifically targeted mRNA iscapable of forming a heteroduplex with the oligonucleotide analogue.

Procedure 3—Nuclease Resistance

A. Evaluation of the Resistance of Oligonucleotide Analogues to Serumand Cytoplasmic Nucleases.

Oligonucleotide analogues of the invention can be assessed for theirresistance to serum nucleases by incubation of the oligonucleotideanalogue in media containing various concentrations of fetal calf serum.Labeled oligonucleotide analogues are incubated for various times,treated with protease K and then analyzed by gel electrophoresis on 20%polyacrylamine-urea denaturing gels and subsequent autoradiography.Autoradiograms are quantitated by laser densitometry. Based upon thelocation of the modified linkage and the known length of theoligonucleotide it is possible to determine the effect on nucleasedegradation by the particular modification. For the cytoplasmicnucleases, an HL 60 cell line can be used. A post-mitochondrialsupernatant is prepared by differential centrifugation and the labelledoligonucleotide analogues are incubated in this supernatant for varioustimes. Following the incubation, the oligonucleotide analogues areassessed for degradation as outlined above for serum nucleolyticdegradation. Autoradiography results are quantitated for comparison ofthe unmodified and the oligonucleotide analogues of the invention.

Table 3 shows the nuclease resistance of certain of the linkages of theinvention to 10% fetal calf serum. As is evident from Table 3, all ofthe linkages tested exhibit greater stability to nucleases of the fetalcalf serum compare to natural nucleotides. In Table 3 the t_(1/2) ofboth the N to N−1 transition and the N−1 to the N−2 transition areshown. In the sequence listing of this table a “*” is used to denote theplace of a linkage of the invention within the sequence and in a likemanner a “p” is used to denote a normal phosphodiester linkage.

TABLE 3 NUCLEASE RESISTANCE (10%) Fetal Calf Serum SEQUENCE = 5′-CpGpApCpTpApTpGpCpApApTpT*TpC -3′ t_(1/2) * BACKBONE N → N-1 → N-2Natural  0.5 hr  1.0 hr N-Methyl-Hydroxyamino  2.0 hr  4.0 hrN-Methyl-Aminohydroxy  5.5 hr 12.5 hr N,N′-Dimethyl-Hydrazino 20.5 hr36.5 hr Aminohydroxy  2.5 hrProcedure 4—5-Lipoxygenase Analysis, Therapeutics and Assays

A. Therapeutics

For therapeutic use, an animal suspected of having a diseasecharacterized by excessive or abnormal supply of 5-lipoxygenase istreated by administering oligonucleotide analogues in accordance withthis invention. Persons of ordinary skill can easily determine optimumdosages, dosing methodologies and repetition rates. Such treatment isgenerally continued until either a cure is effected or a diminution inthe diseased state is achieved. Long term treatment is likely for somediseases.

B. Research Reagents

The oligonucleotide analogues of this invention will also be useful asresearch reagents when used to cleave or otherwise modulate5-lipoxygenase mRNA in crude cell lysates or in partially purified orwholly purified RNA preparations. This application of the invention isaccomplished, for example, by lysing cells by standard methods,optimally extracting the RNA and then treating it with a composition atconcentrations ranging, for instance, from about 100 to about 500 ng per10 Mg of total RNA in a buffer consisting, for example, of 50 mmphosphate, pH ranging from about 4-10 at a temperature from about 30 °toabout 50° C. The cleaved 5-lipoxygenase RNA can be analyzed by agarosegel electrophoresis and hybridization with radiolabeled DNA probes or byother standard methods.

C. Diagnostics

The oligonucleotide analogues of this invention will also be useful indiagnostic applications, Particularly for the determination of theexpression of specific mRNA species in various tissues or the expressionof abnormal or mutant RNA species. In this example, the oligonucleotideanalogues target a hypothetical abnormal mRNA by being designedcomplementary to the abnormal sequence, but would not hybridize to orcleave the normal mRNA.

Tissue samples can be homogenized, and RNA extracted by standardmethods. The crude homogenate or extract can be treated for example toeffect cleavage of the target RNA. The product can then be hybridized toa solid support which contains a bound oligonucleotide complementary toa region on the 5′ side of the cleavage site. Both the normal andabnormal 5′ region of the mRNA would bind to the solid support. The 3′region of the abnormal RNA, which is cleaved by the invention compound,would not be bound to the support and therefore would be separated fromthe normal mRNA.

Targeted mRNA species for modulation relates to 5-lipoxygenase; however,persons of ordinary skill in the art will appreciate that the presentinvention is not so limited and it is generally applicable. Theinhibition or modulation of production of the enzyme 5-lipoxygenase isexpected to have significant therapeutic benefits in the treatment ofdisease. In order to assess the effectiveness of the compositions, anassay or series of assays is required.

D. In Vitro Assays

The cellular assays for 5-lipoxygenase preferably use the humanpromyelocytic leukemia cell line HL-60. These cells can be induced todifferentiate into either a monocyte like cell or neutrophil like cellby various known agents. Treatment of the cells with 1.3% dimethylsulfoxide, DMSO, is known to promote differentiation of the cells intoneutrophils. It has now been found that basal HL-60 cells do notsynthesize detectable levels of 5-lipoxygenase protein or secreteleukotrienes (a downstream product of 5-lipoxygenase). Differentiationof the cells with DMSO causes an appearance of 5-lipoxygenase proteinand leukotriene biosynthesis 48 hours after addition of DMSO. Thusinduction of 5-lipoxygenase protein synthesis can be utilized as a testsystem for analysis of antisense oligonucleotides analogues whichinterfere with 5-lipoxygenase synthesis in these cells.

A second test system for antisense oligonucleotides makes use of thefact that 5-lipoxygenase is a “suicide” enzyme in that it inactivatesitself upon reacting with substrate. Treatment of differentiated HL-60or other cells expressing 5 lipoxygenase, with 10 μM A23187, a calciumionophore, promotes translocation of 5-lipoxygenase from the cytosol tothe membrane with subsequent activation of the enzyme. Followingactivation and several rounds of catalysis, the enzyme becomescatalytically inactive. Thus, treatment of the cells with calciumionophore inactivates endogenous 5-lipoxygenase. It takes the cellsapproximately 24 hours to recover from A23187 treatment as measured bytheir ability to synthesize leukotriene B₄. Oligonucleotide analoguesdirected against 5-lipoxygenase can be tested for activity in two HL-60model systems using the following quantitative assays. The assays aredescribed from the most direct measurement of inhibition of5-lipoxygenase protein synthesis in intact cells to more downstreamevents such as measurement of 5-lipoxygenase activity in intact cells.

The most direct effect which oligonucleotide analogues can exert onintact cells and which can be easily be quantitated is specificinhibition of 5-lipoxygenase protein synthesis. To perform thistechnique, cells can be labelled with ³⁵S-methionine (50 μCi/mL) for 2hours at 37° C. to label newly synthesized protein. Cells are extractedto solubilize total cellular proteins and 5-lipoxygenase isimmunoprecipitated with 5-lipoxygenase antibody followed by elution fromprotein A Sepharose beads. The immunoprecipitated proteins are resolvedby SDS-polyacrylamide gel electrophoresis and exposed forautoradiography. The amount of immunoprecipitated 5-lipoxygenase isquantitated by scanning densitometry.

A predicted result from these experiments would be as follows. Theamount of 5-lipoxygenase protein immunoprecipitated from control cellswould be normalized to 100%. Treatment of the cells with 1 μM, 10 μM,and 30 μM of effective oligonucleotide analogues for 48 hours wouldreduce immunoprecipitated 5-lipoxygenase by 5%, 25% and 75% of control,respectively.

Measurement of 5-lipoxygenase enzyme activity in cellular homogenatescould also be used to quantitate the amount of enzyme present which iscapable of synthesizing leukotrienes. A radiometric assay has now beendeveloped for quantitating 5-lipoxygenase enzyme activity in cellhomogenates using reverse phase HPLC. Cells are broken by sonication ina buffer containing protease inhibitors and EDTA. The cell homogenate iscentrifuged at 10,000×g for 30 min and the supernatants analyzed for5-lipoxygenase activity. Cytosolic proteins are incubated with 10 μM¹⁴C-arachidonic acid, 2 mM ATP, 50 μM free calcium, 100 μg/mlphosphatidylcholine, and 50 mM bis-Tris buffer, pH 7.0, for 5 min at 37°C. The reactions are quenched by the addition of an equal volume ofacetone and the fatty acids extracted with ethyl acetate. The substrateand reaction products are separated by reverse phase HPLC on a NovapakC18 column (Waters Inc., Millford, Mass.). Radioactive peaks aredetected by a Beckman model 171 radiochromatography detector. The amountof arachidonic acid converted into di-HETE's and mono-HETE's is used asa measure of 5-lipoxygenase activity.

A predicted result for treatment of DMSO differentiated HL-60 cells for72 hours with effective oligonucleotide analogues at 1 μM, 10 μM, and 30μM would be as follows. Control cells oxidize 200 pmol arachidonicacid/5 min/10⁶ cells. Cells treated with 1 μM, 10 μM, and 30 μM of aneffective oligonucleotide analogues would oxidize 195 pmol, 140 pmol,and 60 pmol of arachidonic acid/5 min/10⁶ cells respectively.

A quantitative competitive enzyme linked immunosorbant assay (ELISA) forthe measurement of total 5-lipoxygenase protein in cells has beendeveloped. Human 5-lipoxygenase expressed in E. coli and purified byextraction, Q-Sepharose, hydroxyapatite, and reverse phase HPLC is usedas a standard and as the primary antigen to coat microtiter plates. 25ng of purified 5-lipoxygenase is bound to the microtiter platesovernight at 40° C. The wells are blocked for 90 min with 5% goat serumdiluted in 20 mM Tris·HCL buffer, pH 7.4, in the presence of 150 mM NaCl(TBS). Cell extracts (0.2% Triton X-100, 12,000×g for 30 min.) orpurified 5-lipoxygenase were incubated with a 1:4000 dilution of5-lipoxygenase polyclonal antibody in a total volume of 100 μL in themicrotiter wells for 90 min. The antibodies are prepared by immunizingrabbits with purified human recombinant 5-lipoxygenase. The wells arewashed with TBS containing 0.05% tween 20 (TBST), then incubated with100 μL of a 1:1000 dilution of peroxidase conjugated goat anti-rabbitIgG (Cappel Laboratories, Malvern, Pa.) for 60 min at 25° C. The wellsare washed with TBST and the amount of peroxidase labelled secondantibody determined by development with tetramethylbenzidine.

Predicted results from such an assay using a 30 mer oligonucleotideanalogue at 1 μM, 10 μM, and 30 μM would be 30 ng, 18 ng and 5 ng of5-lipoxygenase per 10⁶ cells, respectively with untreated cellscontaining about 34 ng 5-lipoxygenase.

A net effect of inhibition of 5-lipoxygenase biosynthesis is adiminution in the quantities of leukotrienes released from stimulatedcells. DMSO-differentiated HL-60 cells release leukotriene B4 uponstimulation with the calcium ionophore A23187. Leukotriene B4 releasedinto the cell medium can be quantitated by radioimmunoassay usingcommercially available diagnostic kits (New England Nuclear, Boston,Mass.). Leukotriene B4 production can be detected in HL-60 cells 48hours following addition of DMSO to differentiate the cells into aneutrophil-like cell. Cells (2×10⁵ cells/mL) will be treated withincreasing concentrations of oligonucleotide analogues for 48-72 hoursin the presence of 1.3% DMSO. The cells are washed and re-suspended at aconcentration of 2×10⁶ cell/mL in Dulbecco's phosphate buffered salinecontaining 1% delipidated bovine serum albumin. Cells are stimulatedwith 10 μM calcium ionophore A23187 for 15 min and the quantity of LTB4produced from 5×10⁵ cell determined by radioimmunoassay as described bythe manufacturer.

Using this assay the following results would likely be obtained with a15-mer modified linkage bearing antisense oligonucleotide(GCAAGGTCACTGAAG) directed to the 5-LO mRNA. Cells will be treated for72 hours with either 1 μM, 10 μM or 30 μM oligonucleotide analogue inthe presence of 1.3% DMSO. The quantity of LTB₄ produced from 5×10⁵cells would be expected to be about 75 pg, 50 pg, and 35 pg.respectively with untreated differentiated cells producing 75 pg LTB₄.

E. In Vivo Assay

Inhibition of the production of 5-lipoxygenase in the mouse can bedemonstrated in accordance with the following protocol. Topicalapplication of arachidonic acid results in the rapid production ofleukotriene B₄. leukotriene C₄ and prostaglandin E₂ in the skin followedby edema and cellular infiltration. Certain inhibitors of 5-lipoxygenasehave been known to exhibit activity in this assay. For the assay, 2 mgof arachidonic acid is applied to a mouse ear with the contralateral earserving as a control. The polymorphonuclear cell infiltrate is assayedby myeloperoxidase activity in homogenates taken from a biopsy 1 hourfollowing the administration of arachidonic acid. The edematous responseis quantitated by measurement of ear thickness and wet weight of a punchbiopsy. Measurement of leukotriene B₄ produced in biopsy specimens isperformed as a direct measurement of 5-lipoxygenase activity in thetissue. Oligonucleotide analogues will be applied topically to both ears12 to 24 hours prior to administration of arachidonic acid to allowoptimal activity of the compounds. Both ears are pretreated for 24 hourswith either 0.1 μmol, 0.3 μmol, or 1.0 μmol of the oligonucleotideanalogue prior to challenge with arachidonic acid. Values are expressedas the mean for three animals per concentration. Inhibition ofpolymorphonuclear cell infiltration for 0.1 μmol, 0.3 μmol, and 1 μmolis expected to be about 10%, 75% and 92% of control activity,respectively. Inhibition of edema is expected to be about 3%, 58% and90%, respectively while inhibition of leukotriene B₄ production would beexpected to be about 15%, 79% and 99%, respectively.

1. An oligonucleotide analogue in which at least some of the subunits of the analogue have the structure:

wherein B_(x) is a variable base moiety; Q is O, CH₂, CHF or CF₂; X is H; OH; C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl or aralkyl; F; Cl; Br; CN; CF₃; OCF₃; OCN; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; SOCH₃; SO₂CH₃; ONO₂; NO₂; N₃; NH₂; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; or an RNA cleaving group, wherein at least one X is OH; L₁ and L₄ are, independently, CH₂, C═O, C═S, C—NH₂, C—NHR₃, C—OH, C—SH, C—O—R, or C—S—R₁; and L₂ and L₃ are, independently, CR₁R₂, C═CR₁R₂, C═NR₃, P(O)R₄, P(S)R₄, C═O, C═S, O, S, SO, SO₂, NR₃ or SiR₅R₆; or, together, form part of an alkene, alkyne, aromatic ring, carbocycle or heterocycle, or L₁, L₂, L₃ and L₄, together, comprise a —CH═N—NH—CH₂— or —CH₂—O—N═CH— moiety; R₁ and R₂ are, independently, H; OH; SH; NH₂; C₁ to C₁₀ alkyl, substituted alkyl, alkenyl, alkaryl or aralkyl; alkoxy; thioalkoxy; alkylamino; aralkylamino; substituted alkylamino; heterocycloalkyl; heterocycloalkylamino; aminoalkylamino; polyalkylamino; halo; formyl; keto; benzoxy; carboxamido; thiocarboxamido; ester; thioester; carboxamidine; carbamyl; ureido; guanidino; or an RNA cleaving group; R₃ is H, OH, NH₂, lower alkyl, substituted lower alkyl, alkoxy, lower alkenyl, aralkyl, alkylamino, aralkylamino, substituted alkylamino, heterocyclocalkyl, heterocycloalkylamino, aminoalkylamino, polyalkylamino, or an RNA cleaving group; R₄ is OH, SH, NH₂, O-alkyl, S-alkyl, NH-alkyl, O-alkylheterocycle, S-alkylheterocycle, N-alkylheterocycle or a nitrogen-containing heterocycle; and R₅ and R₆ are, independently, C₁ to C₆ alkyl or alkoxy; provided that if L₁ is C═O or C═S then L₂ is not NR₃ or if L₄ is C═O or C═S then L₃ is not NR₃; and that if one of L₂ or L₃ is C═O or C═S then the other of L₂ or L₃ is not NR₃; and if L₂ is P(O)R₄ and R₄ is OH and X is OH and B_(x) is uracil or adenine, then L₃ is not O; that if L₁, L₂ and L₄ are CH₂ and X is H or OH and Q is O then L₃ is not S, SO or SO₂.
 2. The oligonucleotide analogue of claim 1 wherein Q is O.
 3. The oligonucleotide analogue of claim 1 wherein each of L₁ and L₄ are CR₁R₂.
 4. The oligonucleotide analogue of claim 3 wherein R₁ and R₂ are each H.
 5. The oligonucleotide analogue of claim 4 wherein Q is O.
 6. The oligonucleotide analogue of claim 1 wherein L₂ and L₃ are, independently, CR₁R₂, O, P(O)R₄, P(S)R₄ or NR₃.
 7. The oligonucleotide analogue of claim 6 wherein one of L₂ and L₃ is CR₁R₂ and the other of L₂ and L₃ is P(O)R₄ or P(S)R₄.
 8. The oligonucleotide analogue of claim 6 wherein L₂ is O and L₃ is P(O)R₄ or P(S)R₄.
 9. The oligonucleotide analogue of claim 1 wherein each of L₂ and L₃ is NR₃.
 10. The oligonucleotide analogue of claim 9 wherein R₃ is H.
 11. The oligonucleotide analogue of claim 1 wherein L₁ and L₄ are each CH₂ and each of L₂ and L₃ are NR₃.
 12. The oligonucleotide analogue of claim 1 wherein L₂ and L₃ taken together form a portion of a cyclopropyl, cyclobutyl, ethyleneoxy, ethyl aziridine or substituted ethyl aziridine ring.
 13. The oligonucleotide analogue of claim 1 wherein L₂ and L₃ taken together form a portion of a C₃ to C₆ carbocycle or 4-, 5- or 6-membered nitrogen heterocycle.
 14. The oligonucleotide analogue of claim 1 wherein X is H.
 15. The oligonucleotide analogue of claim 1 wherein X is OH.
 16. The oligonucleotide analogue of claim 1 wherein X is H, OH, F, O-alkyl or O-alkenyl and Q is O.
 17. The oligonucleotide analogue of claim 1 wherein B_(x) is adenine, guanine, uracil, thymine, cytosine, 2-aminoadenine or 5-methylcytosine.
 18. The oligonucleotide analogue of claim 17 wherein Q is O.
 19. The oligonucleotide analogue of claim 21 wherein L₁ and L₄ are each CH₂.
 20. The oligonucleotide analogue of claim 19 wherein L₂ and L₃ are each NH.
 21. The oligonucleotide analogue of claim 19 wherein one of L₂ and L₃ is O and the other of L₂ and L₃ is NH.
 22. The oligonucleotide analogue of claim 19 wherein L₂ is NH and L₃ is O.
 23. The oligonucleotide analogue of claim 21 wherein L₂ is O and L₃ is NH.
 24. The oligonucleotide analogue of claim 1 comprising from about 5 to about 50 subunits having said structure.
 25. The oligonucleotide analogue of claim 1 wherein substantially all of the subunits have said structure.
 26. The oligonucleotide analogue of claim 1 wherein substantially alternating subunits have said structure.
 27. The oligonucleotide analogue of claim 1 in a pharmaceutically acceptable carrier.
 28. An oligonucleotide analogue in which at least some of the subunits of the analogue have the structure:

wherein B_(x) is a variable base moiety; Q is O, CH₂, CHF or CF₂; X is H; OH; C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl or aralkyl; F; Cl; Br; CN; CF₃; OCF₃; OCN; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; SOCH₃; SO₂CH₃; ONO₂; NO₂; N₃; NH₂; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; or an RNA cleaving group, wherein at least one X is OH; L₁ and L₄ are, independently, CH₂, C═O, C═S, C—NH₂, C—NHR₃, C—OH, C—SH, C—O—R₁ or C—S—R₁; and L₂ and L₃ are, independently, CR₁R₂, C═CR₁R₂, C═NR₃, P(O)R₄, P(S)R₄, C═O, C═S, O, S, SO, SO₂, NR₃ or SiR₅R₆; or, together, form part of an alkene, alkyne, aromatic ring, carbocycle or heterocycle, or L₁, L₂, L₃ and L₄, together, comprise a —CH═N—NH—CH₂— or —CH₂—O—N═CH— moiety; R₁ and R₂ are, independently, H; OH; SH; NH₂; C₁ to C₁₀ alkyl, substituted alkyl, alkenyl, alkaryl or aralkyl; alkoxy; thioalkoxy; alkylamino; aralkylamino; substituted alkylamino; heterocycloalkyl; heterocycloalkylamino; aminoalkylamino; polyalkylamino; halo; formyl; keto; benzoxy; carboxamido; thiocarboxamido; ester; thioester; carboxamidine; carbamyl; ureido; guanidino; or an RNA cleaving group; R₃ is H, OH, NH₂, lower alkyl, substituted lower alkyl, alkoxy, lower alkenyl, aralkyl, alkylamino, aralkylamino, substituted alkylamino, heterocyclocalkyl, heterocycloalkylamino, aminoalkylamino, polyalkylamino, or an RNA cleaving group; R₄ is OH, SH, NH₂, O-alkyl, S-alkyl, NH-alkyl, O-alkylheterocycle, S-alkylheterocycle, N-alkylheterocycle or a nitrogen-containing heterocycle; and R₅ and R₆ are, independently, C₁ to C₆ alkyl or alkoxy; provided if L₁ is C═O or C═S then L₂ is not NR₃ or if L₄ is C═O or C═S then L₃ is not NR₃; and that if one of L₂ or L₃ is C═O or C═S then the other of L₂ or L₃ is not NR₃; and if L₂ is P(O)R₄ and R₄ is OH and X is OH and B_(x) is uracil or adenine, then L₃ is not O; and that if L₁, L₂ and L₄ are CH₂ and X is H or OH and Q is O then L₃ is not S, SO or SO₂; wherein said oligonucleotide analogue exhibits improved nuclease resistance as compared to corresponding natural oligonucleotides. 